Article

Search-and-replace genome editing without

double-strand breaks or donor DNA

https://doi.org/10.1038/s41586-019-1711-4 Andrew V. Anzalone1,2,3, Peyton B. Randolph1,2,3, Jessie R. Davis1,2,3, Alexander A. Sousa1,2,3,

Luke W. Koblan1,2,3, Jonathan M. Levy1,2,3, Peter J. Chen1,2,3, Christopher Wilson1,2,3,
Received: 26 August 2019
Gregory A. Newby1,2,3, Aditya Raguram1,2,3 & David R. Liu1,2,3*
Accepted: 10 October 2019

Published online: 21 October 2019

Most genetic variants that contribute to disease1 are challenging to correct efficiently
and without excess byproducts2–5. Here we describe prime editing, a versatile and
precise genome editing method that directly writes new genetic information into a
specified DNA site using a catalytically impaired Cas9 endonuclease fused to an
engineered reverse transcriptase, programmed with a prime editing guide RNA
(pegRNA) that both specifies the target site and encodes the desired edit. We
performed more than 175 edits in human cells, including targeted insertions,
deletions, and all 12 types of point mutation, without requiring double-strand breaks
or donor DNA templates. We used prime editing in human cells to correct, efficiently
and with few byproducts, the primary genetic causes of sickle cell disease (requiring a
transversion in HBB) and Tay–Sachs disease (requiring a deletion in HEXA); to install a
protective transversion in PRNP; and to insert various tags and epitopes precisely into
target loci. Four human cell lines and primary post-mitotic mouse cortical neurons
support prime editing with varying efficiencies. Prime editing shows higher or similar
efficiency and fewer byproducts than homology-directed repair, has complementary
strengths and weaknesses compared to base editing, and induces much lower off-
target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing
substantially expands the scope and capabilities of genome editing, and in principle
could correct up to 89% of known genetic variants associated with human diseases.

The ability to make virtually any targeted change in the genome of any
living cell or organism is a longstanding aspiration of the life sciences.
Despite rapid advances in genome editing technologies, the majority
of the more than 75,000 known disease-associated genetic variants in
humans1 remain difficult to correct or install in most therapeutically rel-
evant cell types (Fig. 1a). Programmable nucleases such as CRISPR–Cas9
make double-strand DNA breaks (DSBs) that can disrupt genes by induc-
ing mixtures of insertions and deletions (indels) at target sites2–4. DSBs,
however, are associated with undesired outcomes, including complex
mixtures of products, translocations5, and activation of p536,7. Moreover,
the vast majority of pathogenic alleles arise from specific insertions, dele-
tions, or base substitutions that require more precise editing technolo-
gies to correct (Fig. 1a, Supplementary Discussion). Homology-directed
repair (HDR) stimulated by DSBs8 has been widely used to install precise
DNA changes. HDR, however, relies on exogenous donor DNA repair tem-
plates, typically generates an excess of indels from end-joining repair of
DSBs, and is inefficient in most therapeutically relevant cell types (T cells
and some types of stem cell being important exceptions)9,10. Whereas
enhancing the efficiency and precision of DSB-mediated editing remains
the focus of promising efforts11–15, these challenges motivate the explora-
tion of alternative precision genome editing strategies.
Base editing can efficiently install the four transition mutations (C→T,
G→A, A→G, and T→C) without requiring DSBs in many cell types and
organisms, including mammals16–19, but cannot currently perform the
eight transversion mutations (C→A, C→G, G→C, G→T, A→C, A→T, T→A,
and T→G), such as the T•A-to-A•T mutation needed to directly correct
the most common cause of sickle cell disease (HBB(E6V)). In addition,
no DSB-free method has been reported to perform targeted deletions,
such as the removal of the four-base duplication that causes Tay-Sachs
disease (HEXA1278+TATC), or targeted insertions, such as the three-base
insertion required to directly correct the most common cause of cystic
fibrosis (CFTR(ΔF508)). Targeted transversions, insertions, and dele-
tions are therefore difficult to install or correct efficiently and without
excess byproducts in most cell types, even though they collectively
account for most known pathogenic alleles (Fig. 1a).
Here we describe the development of prime editing, a ‘search-and-
replace’ genome editing technology that mediates targeted insertions,
deletions, all 12 possible base-to-base conversions, and combinations
thereof in human cells without requiring DSBs or donor DNA templates.
Prime editors (PEs), initially exemplified by PE1, use a reverse transcriptase
(RT) fused to an RNA-programmable nickase and a prime editing guide
RNA (pegRNA) to copy genetic information directly from an extension
on the pegRNA into the target genomic locus. PE2 uses an engineered RT
to increase editing efficiencies, while PE3 nicks the non-edited strand to
induce its replacement and further increase editing efficiency, typically
to 20–50% with 1–10% indel formation in human HEK293T cells. Prime

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA. 2Department of Chemistry and Chemical Biology, Harvard University,
1

Cambridge, MA, USA. 3Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA. *e-mail: drliu@fas.harvard.edu

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Article
a Known human pathogenic genetic variants b Prime editor (PE) and pegRNA
Target DNA
2.0% Reverse pegRNA nick site
2.5%
3.6% 0.9% transcriptase Protospacer PAM
domain
6.8% Transition point mutation 5′• • • • • • 3′
Transversion point mutation
pegRNA 3′ 3′• • • • • • 5′
Deletion
Duplication Edit position: +1 +4 +12
30%
8.2% Copy number loss Prime editing range
Copy number gain for SpCas9 PEs
Insertion Prime editing scope:
Insertion and deletion 5′ All 4 transition point mutations
26% 20% Other All 8 transversion point mutations
Insertions (1 bp to ≥ 44 bp)
Cas9 nickase Deletions (1 bp to ≥ 80 bp)
domain Combinations of the above
Total variants = 75,122

Primer- RT template Direct polymerization of edited

binding site including edit DNA from the pegRNA into
c the target site
3′ 3’ 3′
3’
PE +
5′ L
pegRNA 5′•••
+ DNA Nicking 3′••• Hybridization of Reverse
of PAM primer-binding transcription
strand 5′ site in pegRNA
to PAM strand

3′ Flap 5′ Flap
(contains edit) (lacks edit)
3’ Flap 5′ 5′ Flap DNA
equilibration Edited DNA
cleavage repair
5′••• ••• 3′ 5′••• ••• 3′ 5′••• ••• 3′ 5′••• ••• 3′
3′••• ••• 5′ 3′••• ••• 5′ 3′••• ••• 5′ 3′••• ••• 5′
Ligation

f 3′-extended pegRNA 5′-extended pegRNA

d 5′-extended pegRNAs e 3′-extended pegRNAs stop codon correction stop codon correction
T•A-to-A•T transversion T•A-to-A•T transversion
Non-nicked
56 nt DNA substrate (51 nt)
RT templated RT
49 nt products
products product
42 nt (51 nt)
Pre-nicked
Pre-nicked substrate (34 nt)
substrate
(34 nt) 3′-extended pegRNA 3′-extended pegRNA
frameshift correction frameshift correction
Pre-nicked dsDNA Pre-nicked dsDNA + – + + – 1-nt insertion 1-nt deletion
+ + + + + + +
dCas9 + + + + – – – Non-nicked dsDNA – + – – +
RT + + + + + + + dCas9 – – + – –
sgRNA + – – – – – – Cas9 H840A nickase – – – + +
5′-extended pegRNA – + + + + + + RT – – + + +
3′-extended pegRNA – – + + +
Length of pegRNA – 8 15 22 8 15 22
RT template (nt)

Fig. 1 | Overview of prime editing and feasibility studies in vitro and in yeast
cells. a, The 75,122 known pathogenic human genetic variants in ClinVar
(accessed July, 2019), classified by type. b, A prime editing complex consists of
a PE protein containing an RNA-guided DNA-nicking domain, such as Cas9
nickase, fused to an RT domain and complexed with a pegRNA. The PE–pegRNA
complex enables a variety of precise DNA edits at a wide range of positions.
spCas9, Streptococcus pyogenes Cas9. c, The PE–pegRNA complex binds the
target DNA and nicks the PAM-containing strand. The resulting 3′ end
hybridizes to the PBS, then primes reverse transcription of new DNA containing
the desired edit using the RT template of the pegRNA. Equilibration between
the edited 3′ flap and the unedited 5′ flap, cellular 5′ flap cleavage and ligation,
and DNA repair results in stably edited DNA. d, In vitro primer extension assays
with 5′-extended pegRNAs, pre-nicked dsDNA substrates containing 5′-Cy5-
labelled PAM strands, dCas9, and a commercial M-MLV RT variant (RT,
Superscript III). dCas9 was complexed with pegRNAs, then added to DNA
substrates along with the indicated components. After 1 h, reactions were
analysed by denaturing PAGE to visualize Cy5 fluorescence. e, Primer extension
assays performed as in d using 3′-extended pegRNAs pre-complexed with
dCas9 or Cas9(H840A) nickase, and pre-nicked or non-nicked dsDNA
substrates. f, Yeast colonies transformed with GFP–mCherry fusion reporter
plasmids edited in vitro with pegRNAs, Cas9 nickase, and RT. Plasmids
containing nonsense or frameshift mutations between GFP and mCherry were
edited with pegRNAs that restored mCherry translation via transversion, 1-bp
insertion, or 1-bp deletion. GFP and mCherry double-positive cells (yellow)
reflect successful editing. Images in d–f are representative of n = 2 independent
replicates. For gel source data, see Supplementary Fig. 1.

editing offers much lower off-target activity than Cas9 at known Cas9 off-
target loci, far fewer byproducts and higher or similar efficiency compared
to Cas9-initiated HDR, and complementary strengths and weaknesses
compared to base editors. By enabling precise targeted insertions, dele-
tions, and all 12 possible classes of point mutations without requiring DSBs
or donor DNA templates, prime editing has the potential to advance the
study and correction of the vast majority of pathogenic alleles.
Prime editing strategy
Cas9 targets DNA using a guide RNA containing a spacer sequence that
hybridizes to the target DNA site2–4,20,21. We envisioned the generation of
guide RNAs that both specify the DNA target and contain new genetic
information that replaces target DNA nucleotides. To transfer informa-
tion from these engineered guide RNAs to target DNA, we proposed that
genomic DNA, nicked at the target site to expose a 3′-hydroxyl group,
could be used to prime the reverse transcription of an edit-encoding
extension on the engineered guide RNA (the pegRNA) directly into the
target site (Fig. 1b, c, Supplementary Discussion).
These initial steps result in a branched intermediate with two redun-
dant single-stranded DNA flaps: a 5′ flap that contains the unedited DNA
sequence and a 3′ flap that contains the edited sequence copied from
the pegRNA (Fig. 1c). Although hybridization of the perfectly comple-
mentary 5′ flap to the unedited strand is likely to be thermodynamically

2 | Nature | www.nature.com
 a HEK3, 10-nt RT template EMX1, 13-nt RT template FANCF, 17-nt RT template RNF2, 11-nt RT template HEK4, 13-nt RT template HEK3, 13-nt PBS,
+1 T to A +5 G to T +5 G to T +1 C to A +2 G to T 10-nt RT template
with the specified edit (%)
Total sequencing reads

10 PE1 15 PE1 20 PE1 5 PE1 10 PE1 30

PE1
PE2 PE2 PE2 PE2 PE2 25 PE2
8 4 8
15
10 20
6 3 6
10 15
4 2 4
5 10
5
2 1 2 5
0 0 0 0 0 0
8 9 10 11 12 13 14 15 16 17 9 10 11 12 13 14 15 16 17 8 9 10 11 12 13 14 15 16 17 9 10 11 12 13 14 15 16 17 7 8 9 10 11 12 13 14 15 +1 T +1 A +1 CTT
PBS length (nt) PBS length (nt) PBS length (nt) PBS length (nt) PBS length (nt) del ins ins

b
PE2, 13-nt PBS PE2, 13-nt PBS PE2, 13-nt PBS PE2, 15-nt PBS PE2, 11-nt PBS
with the specified edit (%)

25 EMX1, +1 G to C
Total sequencing reads

20 HEK3, +1 T to A 30 FANCF, +5 G to T 15 RNF2, +1 C to A 20 HEK4, +2 G to T

20
15 15
15 20 10
10 10
10
10 5
5 5 5

0 0 0 0 0
10 11 12 13 14 15 16 17 18 19 20 10 11 12 13 14 15 16 17 18 19 20 10 11 12 13 14 15 16 17 18 19 20 10 11 12 13 14 15 16 17 18 19 20 7 8 9 10 1112 13 14 15 16 17 18 19 20
RT template length (nt) RT template length (nt) RT template length (nt) RT template length (nt) RT template length (nt)

Fig. 2 | Prime editing of genomic DNA in human cells by PE1 and PE2. a, Use of b, PE2 editing efficiencies with varying RT template lengths at five genomic
an engineered M-MLV reverse transcriptase (D200N, L603W, T306K, W313F, sites in HEK293T cells. Editing efficiencies reflect sequencing reads that
T330P) in PE2 substantially improves prime editing efficiencies at five genomic contain the intended edit and do not contain indels among all treated cells,
sites in HEK293T cells, and small insertion and small deletion edits at HEK3. with no sorting. Mean ± s.d. of n = 3 independent biological replicates.

favoured, 5′ flaps are the preferred substrate for structure-specific a conventional sgRNA (Fig. 1d). These results demonstrate that nicked
endonucleases such as FEN122, which excises 5′ flaps generated during DNA exposed by dCas9 is competent to prime reverse transcription
lagging-strand DNA synthesis and long-patch base excision repair. The from a pegRNA.
redundant unedited DNA may also be removed by 5′ exonucleases Next, we tested non-nicked dsDNA substrates with a Cas9(H840A)
such as EXO123. nickase that nicks the PAM-containing strand2. In these reactions,
We reasoned that preferential 5′ flap excision and 3′ flap ligation 5′-extended pegRNAs generated reverse transcription products inef-
could drive the incorporation of the edited DNA strand, creating ficiently (Extended Data Fig. 1f), but 3′-extended pegRNAs enabled
heteroduplex DNA containing one edited strand and one unedited efficient Cas9 nicking and reverse transcription (Fig. 1e). The use of
strand (Fig. 1c). DNA repair to resolve the heteroduplex by copying the 3′-extended pegRNAs generated only a single apparent product, despite
information in the edited strand to the complementary strand would the theoretical possibility that reverse transcription could terminate
permanently install the edit (Fig. 1c). On the basis of a similar strategy anywhere within the pegRNA. DNA sequencing of reactions with Cas9
we developed to favourably resolve heteroduplex DNA during base nickase, RT, and 3′-extended pegRNAs revealed that the complete RT
editing16–18, we hypothesized that nicking the non-edited DNA strand template sequence was reverse transcribed into the DNA substrate
might bias DNA repair to preferentially replace the non-edited strand. (Extended Data Fig. 1g). These experiments establish that 3′-extended
pegRNAs can direct Cas9 nickase and template reverse transcription
in vitro.
Validation in vitro and in yeast To evaluate the eukaryotic cell DNA repair outcomes of 3′ flaps
First, we tested whether the 3′ end of the protospacer-adjacent motif produced by pegRNA-programmed reverse transcription in vitro, we
(PAM)-containing DNA strand cleaved by the RuvC nuclease domain performed in vitro prime editing on reporter plasmids, then trans-
of Cas9 was sufficiently accessible to prime reverse transcription. We formed the reaction products into yeast cells (Extended Data Fig. 2).
designed pegRNAs by adding to single guide RNAs (sgRNAs) a primer We constructed reporter plasmids encoding EGFP and mCherry sepa-
binding site (PBS) that allows the 3′ end of the nicked DNA strand to rated by a linker containing an in-frame stop codon, +1 frameshift, or
hybridize to the pegRNA, and an RT template containing the desired −1 frameshift. When plasmids were edited in vitro with Cas9 nickase,
edit (Fig. 1c). We constructed candidate pegRNAs by extending sgR- RT, and 3′-extended pegRNAs encoding a transversion that corrects
NAs on either end with a PBS sequence (5–6 nucleotides (nt)) and an the premature stop codon, 37% of yeast transformants expressed both
RT template (7–22 nt), and confirmed that 5′-extended pegRNAs sup- GFP and mCherry (Fig. 1f, Extended Data Fig. 2). Reactions edited with
port Cas9 binding to target DNA in vitro and that both 5′-extended and 5′-extended pegRNAs yielded fewer GFP and mCherry double-positive
3′-extended pegRNAs support Cas9-mediated DNA nicking in vitro colonies (9%). Productive editing was also observed using 3′-extended
and DNA cleavage in mammalian cells (Extended Data Fig. 1a–c). Next, pegRNAs that insert a single nucleotide (15%) or delete a single nucle-
we tested the compatibility of these candidate pegRNAs with reverse otide (29%) to correct frameshift mutations (Fig. 1f, Extended Data
transcription using pre-nicked 5′-Cy5-labelled double-stranded DNA Fig. 2). These results demonstrate that DNA repair in eukaryotic cells
(dsDNA) substrates, catalytically dead Cas9 (dCas9), and a commercial can resolve 3′ DNA flaps from prime editing to incorporate precise
Moloney murine leukaemia virus (M-MLV) RT variant (Extended Data transversions, insertions, and deletions.
Fig. 1d). When all components were present, the labelled DNA strand
was efficiently converted into longer DNA products with gel mobilities
consistent with reverse transcription along the RT template (Fig. 1d, Prime editor 1
Extended Data Fig. 1d, e). Omission of dCas9 led to nick translation Encouraged by these observations, we sought to develop a prime edit-
products that resulted from RT-mediated DNA polymerization on the ing system with a minimum number of components that could edit
DNA template, with no pegRNA information transfer. No DNA polym- genomic DNA in mammalian cells. We transfected HEK293T cells with
erization products were observed when the pegRNA was replaced by one plasmid encoding a fusion of the wild-type M-MLV RT through a

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Article
a Prime editor c RNF2

the specified edit or indels (%)

and pegRNA

Total sequencing reads with

60
encoding Y 3′ Flap excision Ligation Unchanged sequence Correct edit (without indels)
X′ X X′X Y X′X X′ X (remains a substrate for Indels
3′ subsequent prime editing)
Starting DNA 40
sequence Disfavoured repair
Flap of the edited strand
PE3 and PE3b
equilibration
X = Original DNA sequence use an sgRNA Preferential repair 20
X′ = Complement of X 5′ to nick the of the nicked
5′ Flap excision non-edited strand non-edited strand Desired
Y = Edited DNA sequence X′ Y X′ Y Y′ Y prime editing
X X′ Y
Y′ = Complement of Y Ligation outcome 0

PE2
PE3
+41 nick
PE3b
+5 nick
PE2
PE3
+41 nick
PE3b
+5 nick
pegRNA nick site
b PAM RNF2 +1 C•G to A•T (without indels)
5′••• ••• 3′ Edited strand
EMX1 +5 G•C to T•A (without indels) +4 A to C +4 A to G
3′••• ••• 5′ Complementary strand
60 FANCF +5 G•C to T•A (without indels)

with the specified edit or indels

FANCF
the specified edit or indels (%)

–10 –1 +1 +10
Total sequencing reads with

60

% of total sequencing reads

HEK3 +2 G•C to C•G (without indels)
50 Complementary-strand nick location HEK4 +2 G•C to T•A (without indels) Correct edit
Indels (without indels)
40 40 Indels

30
20
20

10
0

PE2
PE3
+48 nick
PE3b
+7 nick
PE2

PE3
+48 nick
PE3b
+7 nick
0
Complementary-
None
+41
+67
None
–116
–57
+14
+27
+53
+80
None
–78
–50
–27
–17
+21
+48
None
–108
–38
+26
+37
+63
+90
None
–95
–52
–26
+52
+74
strand nick location

RNF2 EMX1 FANCF HEK3 HEK4 +5 G to T +7 A to C

Fig. 3 | PE3 and PE3b systems nick the non-edited strand to increase prime
editing efficiency. a, Overview of prime editing by PE3. After initial synthesis
of the edited strand, 5′ flap excision leaves behind a DNA heteroduplex
containing one edited strand and one non-edited strand. Mismatch repair
resolves the heteroduplex to give either edited or non-edited products.
Nicking the non-edited strand favours repair of that strand, resulting in
preferential generation of duplex DNA containing the desired edit. b, The
effect of complementary strand nicking on prime editing efficiency and indel
formation. ‘None’ refers to PE2 controls, which do not nick the complementary
strand. c, Comparison of editing efficiencies with PE2, PE3, and PE3b (edit-
specific complementary strand nick). Editing efficiencies reflect sequencing
reads that contain the intended edit and do not contain indels among all
treated cells, with no sorting. Mean ± s.d. of n = 3 independent biological
replicates.

flexible linker to either terminus of the Cas9(H840A) nickase, and a
second plasmid encoding a pegRNA (Extended Data Fig. 3a). Initial
attempts led to no detectable editing.
Extension of the PBS in the pegRNA to 8–15 bases, however, led to
detectable installation of a transversion at the HEK293 site 3 (hereafter
referred to as HEK3) target site, with higher efficiencies when the RT was
fused to the C terminus of Cas9 nickase than when it was fused to the N
terminus (Extended Data Fig. 3b). These results suggest that wild-type
M-MLV RT fused to Cas9 requires longer PBS sequences for genome
editing in human cells compared to what is required in vitro using the
commercial variant of M-MLV RT supplied in trans. We designated
this M-MLV RT fused to the C terminus of Cas9(H840A) nickase as PE1.
We tested the ability of PE1 to introduce transversion point mutations
at four additional genomic sites specified by the pegRNA (Fig. 2a).
Editing efficiency at these sites was dependent on PBS length, with
maximal editing efficiencies reaching 0.7–5.5% (Fig. 2a). Indels from PE1
were minimal, averaging 0.2 ± 0.1% (mean ± s.d.) for the five sites under
conditions that maximized each site’s editing efficiency (Extended
Data Fig. 3a–f). PE1 also mediated targeted insertions and deletions
with 4–17% efficiency at the HEK3 locus (Fig. 2a). These findings show
that PE1 can directly install targeted transversions, insertions, and
deletions without requiring DSBs or DNA templates.
Prime editor 2
We hypothesized that engineering the RT in PE1 might improve the
efficiency of DNA synthesis during prime editing. M-MLV RT muta-
tions that increase thermostability24,25, processivity24, and DNA–RNA
substrate affinity26, and that inactivate RNaseH activity27, have been
reported. We constructed 19 variants of PE1 containing a variety of RT
mutations to evaluate their editing efficiency in human cells.
First, we investigated M-MLV RT variants that support reverse tran-
scription at elevated temperatures24. Introduction of D200N, L603W
and T330P into M-MLV RT, hereafter referred to as M3, led to a 6.8-
fold average increase in transversion and insertion editing efficiency
across five genomic loci in HEK293T cells compared to PE1 (Extended
Data Fig. 4).
We tested additional RT mutations that have been shown to enhance
binding to the template–PBS complex, enzyme processivity, and ther-
mostability26. Among the 14 additional mutants analysed, addition of
T306K and W313F to M3 improved editing efficiency an additional
1.3-fold to 3.0-fold for six transversion or insertion edits across five
genomic sites (Extended Data Fig. 4). This pentamutant RT incor-
porated into PE1 (Cas9(H840A)–M-MLV RT(D200N/L603W/T330P/
T306K/W313F)) is hereafter referred to as prime editor 2 (PE2).
PE2 installs single-nucleotide transversion, insertion, and dele-
tion mutations with substantially higher efficiency than PE1, and is
compatible with shorter PBS sequences, consistent with enhanced
engagement of transient genomic DNA–PBS complexes (Fig. 2a). On
average, PE2 led to a 1.6- to 5.1-fold improvement in the efficiency of
prime editing point mutations over PE1. PE2 also performed targeted
insertions and deletions more efficiently than PE1 (Fig. 2a, Extended
Data Fig. 4d).
Optimization of pegRNAs
We systematically probed the relationship between pegRNA structure
and PE2 editing efficiency. Priming regions with lower G/C content
generally required longer PBS sequences, consistent with the energetic
requirements of hybridization of the nicked DNA strand to the pegRNA
PBS (Fig. 2a). No PBS length or G/C content level was strictly predictive
of editing efficiency, suggesting that other factors such as DNA primer
or RT template secondary structure also influence editing activity.
We recommend starting with a PBS length of about 13 nt, and testing
different PBS lengths during optimization, especially if the priming
region deviates from about 40–60% G/C content.

4 | Nature | www.nature.com
 a PE3, 10-nt RT template, b PE3, 34-nt RT template, c PE3, 14-nt RT template,
nicking at +90, HEK3 nicking at +90, HEK3 nicking at +41, RNF2

targeted point mutation or indels (%)

targeted point mutation or indels (%)

targeted point mutation or indels (%)
60 60 60

Total sequencing reads with

Total sequencing reads with

Correct edit (without indels) Correct edit (without indels) Correct edit (without indels)

Total sequencing reads with

Indels 50 Indels 50 Indels
50
40 40 40

30 30 30

20 20 20

10 10 10

0 0 0

+1 T to C
+1 T to G

+2 G to C
+2 G to T
+3 A to C
+3 A to G
+3 A to T

+4 T to C
+4 T to G

+5 G to C
+5 G to T

+6 G to C
+6 G to T

+7 C to G
+7 C to T
+8 A to C
+8 A to G
+8 A to T

+12 G to C
+14 A to T
+17 G to C
+20 G to C
+23 C to G

+26 C to G
+30 C to G
+ 33 C to G

+1 C to G
+1 C to T

+2 T to G
+3 G to C
+4 A to C
+4 A to T
+4 A to G
+5 G to T

+7 T to C
+1 T to A

+2 G to A

+4 T to A

+5 G to A

+6 G to A

+7 C to A

+1 T to A

+24 T to A

+1 C to A

+2 T to A

+6 G to A
d PE3, 15-nt RT template, e PE3, 22-nt RT template, g PE3, larger deletions,
nicking at +38, RUNX1 nicking at +57, VEGFA
targeted point mutation or indels (%)

targeted point mutation or indels (%)

nicking at +90, HEK3

targeted deletion or indels (%)

50 Correct edit (without indels) 70 Correct edit (without indels) 100 Correct edit (without indels)

Total sequencing reads with

Total sequencing reads with

Total sequencing reads with

Indels Indels Indels
60
40 80
50
30 40 60

20 30 40
20
10 20
10
0 0 0
+1 C to G
+1 C to T

+3 A to C
+3 A to G
+3 A to T

+4 T to C
+4 T to G
+5 G to T
+6 G to C

+1 T to C
+1 T to G

+3 A to C
+3 A to G
+3 A to T
+5 G to T
+6 G to C

+7 C to T
+9 C to G

del +1–5

del +1–10

del +1–15

del +1–25

del +1–30

del +1–80
+1 C to A

+2 G to A

+4 T to A

+1 T to A

+2 G to A

+7 C to A
f PE3, small insertions and small deletions h PE3, combination edits
the specified edit or indels (%)

the specified edit or indels (%)

70 100
Total sequencing reads with

Total sequencing reads with

Correct edit (without indels) Correct edit (without indels)
Indels Indels
60
80
50
40 60

30 40
20
20
10
0 0
+1 A ins
+1 CTT ins
+1 T del
+1–3 TGA del

+1 T ins
+1 GTA ins
+4 A del
+3–5 GAG del

+3 C ins
+4 GAT ins
+6 G del
+5–7 GGA del

+6 T ins
+1 TGC ins
+5 G del
+4–6 GGG del

+1 C ins
+1 ATG ins
+2 G del
+2–4 GAT del

+4 C ins
+2 ACA ins
+3 A del
+2–4 GAG del

+4 C ins
+1 TCA ins
+3 A del
+3–5 AGG del

+1 CTT ins
and +5 G del
+1 CTT ins
and +2 G to C
+1 T del and
+5 G to C
+2 G to C and
+6 G to T
+2 AA ins and
+3–4 GA del
+1 A ins and
+5 G to C
+1–2 CT del
and +6 G to T
+1 C to A and
+5 G to T
+1 T ins and
+4–5 TG del
+1 T ins and
+6 G to A
+2 C del and
+5 G to T
+1 A to C and
+6 G to T
HEK3 RNF2 FANCF EMX1 RUNX1 VEGFA DNMT1 HEK3 RNF2 FANCF

Fig. 4 | Targeted insertions, deletions, and all 12 types of point mutation (c), RUNX1 (d), and VEGFA (e). f, Targeted 1- and 3-bp insertions, and 1- and 3-bp
with PE3 at seven endogenous genomic loci in HEK293T cells. a, All 12 types deletions with PE3 at seven endogenous genomic loci. g, Targeted precise
of single-nucleotide edit from position +1 to +8 of the HEK3 site using a 10-nt RT deletions of 5–80 bp at HEK3. h, Combination edits at three endogenous
template, counting the first nucleotide following the pegRNA-induced nick as genomic loci. Editing efficiencies reflect sequencing reads that contain the
position +1. b, Long-range PE3 edits at HEK3 using a 34-nt RT template. c–e, PE3- intended edit and do not contain indels among all treated cells, with no sorting.
mediated transition and transversion edits at the specified positions for RNF2 Mean ± s.d. of n = 3 independent biological replicates.

Next, we systematically evaluated pegRNAs with RT templates nickase already present in PE2 and a simple sgRNA (Fig. 3a). As the
10–20 nt long at five genomic target sites using PE2 (Fig. 2b), and with RT edited DNA strand is also nicked to initiate prime editing, we tested a
templates up to 31 nt at three genomic sites (Extended Data Fig. 5a–c). variety of nick locations on the non-edited strand to minimize DSBs
As with PBS length, RT template length could also be varied to maxi- that lead to indels.
mize prime editing efficiency, although many RT template lengths of We first tested this strategy, designated PE3, at five genomic sites in
ten or more nucleotides performed comparably. As some target sites HEK293T cells using sgRNAs that induce nicks 14–116 nt away from the
preferred longer RT templates (more than 15 nt; FANCF, EMX1), whereas site of the pegRNA-induced nick. In four of the five sites tested, nicking
other loci preferred shorter RT templates (HEK3 and HEK293 site 4, the non-edited strand increased editing efficiency by 1.5- to 4.2-fold
hereafter referred to as HEK4) (Fig. 2b), we recommend starting with compared to PE2, to as high as 55% (Fig. 3b). Although the optimal
about 10–16 nt and testing shorter and longer RT templates during nicking position varied depending on the genomic site (Supplementary
pegRNA optimization. Discussion), nicks positioned 3′ of the edit about 40–90 bp from the
Notably, the use of RT templates that place a C adjacent to the 3′ hair- pegRNA-induced nick generally increased editing efficiency (averaging
pin of the sgRNA scaffold generally resulted in lower editing efficiency 41%) without excess indel formation (6.8% average indels for the sgRNA
(Extended Data Fig. 5a–c). We speculate that a C as the first nucleotide with the highest editing efficiency) (Fig. 3b). We recommend starting
of the 3′ extension can disrupt guide RNA structure by pairing with G81, with non-edited strand nicks about 50 bp from the pegRNA-mediated
which normally forms a pi stack with Y1356 in Cas9 and a non-canonical nick, and testing alternative nick locations if indel frequencies exceed
base pair with A68 of the sgRNA28. Because many RT template lengths acceptable levels.
support prime editing, we recommend designing pegRNAs so that the Nicking the non-edited strand only after resolution of the edited
first base of the 3′ extension is not C. strand flap should minimize the presence of concurrent nicks, thereby
minimizing formation of DSBs and indels. To achieve this goal, we
designed sgRNAs with spacers that matched the edited strand, but
Prime editor 3 systems not the original allele. Using this strategy, denoted PE3b, mismatches
The resolution of heteroduplex DNA from PE2 containing one edited between the spacer and the unedited allele should disfavour sgRNA
and one non-edited strand determines long-term editing outcomes. nicking until after editing of the PAM strand has taken place. PE3b
To optimize base editing we previously used Cas9 nickase to nick the resulted in a 13-fold decrease in the average number of indels (0.74%)
non-edited strand, directing DNA repair to that strand using the edited compared to PE3, without any evident decrease in editing efficiency
strand as a template16–18. To apply this strategy to enhance prime edit- (Fig. 3c). When the edit lies within a second protospacer, we recom-
ing, we tested nicking the non-edited strand using the Cas9(H840A) mend the PE3b approach.

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Together, these findings establish that PE3 systems improve editing
efficiencies about threefold compared with PE2, albeit with a higher
range of indels than PE2. When it is possible to nick the non-edited
strand with an sgRNA that requires editing before nicking, the PE3b sys-
tem offers PE3-like editing levels while greatly reducing indel formation.
To demonstrate the targeting scope and versatility of prime editing
with PE3, we performed all 24 possible single-nucleotide substitutions
across the +1 to +8 positions (counting the first base 3′ of the pegRNA-
induced nick as position +1) of the HEK3 target site using PE3 and pegR-
NAs with 10-nt RT templates (Fig. 4a). These 24 edits collectively cover
all 12 possible transition and transversion mutations, and proceeded
with average editing efficiencies (containing no indels) of 33 ± 7.9%,
with 7.5 ± 1.8% average indels.
Notably, long-distance RT templates can also give rise to efficient
prime editing. Using PE3 with a 34-nt RT template, we installed point
mutations at positions +12, +14, +17, +20, +23, +24, +26, +30, and +33 in
the HEK3 locus with 36 ± 8.7% average efficiency and 8.6 ± 2.0% indels
(Fig. 4b). Other RT templates of 30 or more nucleotides at three other
genomic sites also supported prime editing (Extended Data Fig. 5a–c).
As an NGG PAM on either DNA strand occurs on average every 8 bp, far
less than edit-to-PAM distances that support efficient prime editing,
prime editing is not substantially constrained by the availability of a
nearby PAM sequence, in contrast to other precision editing meth-
ods11,15,16. Given the presumed relationship between RNA secondary
structure and prime editing efficiency, when designing pegRNAs for
long-range edits we recommend testing RT templates of various lengths
and, if necessary, sequence compositions (for example, using synony-
mous codons).
To further test the scope and limitations of PE3 for introducing point
mutations, we tested 72 additional edits covering all possible types of
point mutation across six additional genomic target sites (Fig. 4c–e,
Extended Data Fig. 5d–f). Editing efficiency averaged 25 ± 14%, while
indel formation averaged 8.3 ± 7.5%. Because the pegRNA RT template
includes the PAM sequence, prime editing can induce changes in the
PAM sequence. In these cases, we observed higher editing efficiency
(averaging 39 ± 9.7%) and lower indel generation (averaging 5.0 ± 2.9%;
Fig. 4, mutations at +5 or +6), potentially due to the inability of Cas9
nickase to re-bind and nick the edited strand before the repair of the
complementary strand. We recommend editing the PAM, in addition
to other desired changes, whenever possible.
Next, we performed 28 targeted small insertions and small deletions
at seven genomic sites using PE3 (Fig. 4f). Targeted 1-bp and 3-bp inser-
tions proceeded with an average efficiency of 32 ± 9.8% and 39 ± 16%,
respectively. Targeted 1-bp and 3-bp deletions were also efficient,
averaging 29 ± 14% and 32 ± 11% editing, respectively. Indel generation
(beyond the target insertion or deletion) averaged 6.8 ± 5.4%. Because
insertions and deletions between positions +1 and +6 alter the location
or structure of the PAM, we speculate that insertions or deletions at
these positions are more efficient because they prevent re-engagement
of the edited strand.
We also tested PE3 for its ability to mediate larger precise deletions
of 5–80 bp at the HEK3 site (Fig. 4g). We observed very high editing
efficiencies (52–78%) for precise 5-, 10-, 15-, 25-, and 80-bp deletions,
with indels averaging 11 ± 4.8%. Finally, we tested the ability of PE3 to
mediate 12 combinations of insertions, deletions, and/or point muta-
tions across three genomic sites. These combination edits were also
very efficient, averaging 55% editing with 6.4% indels (Fig. 4h). Together,
the 156 distinct edits in Fig. 4 and Extended Data Fig. 5d–f establish the
versatility, precision, and targeting flexibility of PE3 systems.
Prime editing compared with base editing
Cytidine base editors (CBEs) and adenine base editors (ABEs) can install
transition mutations efficiently and with few indels16–18. The applica-
tion of base editing can be limited by unwanted bystander edits from
6 | Nature | www.nature.com
the presence of multiple cytidine or adenine bases within the base
editing activity window16–18,29, or by the absence of a PAM positioned
about 15 ± 2 nt from the target nucleotide16,30. We anticipated that prime
editing could complement base editing when bystander edits are unac-
ceptable or when the target site lacks a suitably positioned PAM.
We compared PEs and CBEs at three genomic loci that contain multi-
ple target cytosines in the canonical base editing window (protospacer
positions 4–8, counting the PAM as positions 21–23) using current-gen-
eration CBEs31 without or with nickase activity (BE2max and BE4max,
respectively), or using analogous PE2 and PE3 prime editing systems.
Among the nine total cytosines within the base editing windows of the
three sites, BE4max yielded 2.2-fold higher average total C•G-to-T•A
conversion than PE3 for bases in the centre of the base editing win-
dow (protospacer positions 5–7, Extended Data Fig. 6a). However, PE3
outperformed BE4max by 2.7-fold at cytosines positioned outside the
centre of the base editing window. Overall, indel frequencies for PE2
were very low (averaging 0.86 ± 0.47%), and for PE3 were similar to or
modestly higher than that of BE4max (PE3: 2.5–21%; BE4max: 2.5–14%)
(Extended Data Fig. 6b).
For the installation of precise edits (with no bystander editing), the
efficiency of prime editing greatly exceeded that of base editing at
the above sites, which, like most genomic DNA sites, contain multi-
ple cytosines within the base editing window. BE4max generated few
products containing only the single target base-pair conversion with
no bystander edits. By contrast, prime editing at this site could be used
to selectively install a C•G-to-T•A edit at any position or combination
of positions (Extended Data Fig. 6c).
We also compared nicking and non-nicking adenine base editors
(ABEs) with PE3 and PE2, with similar results (Extended Data Fig. 6d–f,
Supplementary Discussion). Collectively, these results indicate that
base editing and prime editing offer complementary strengths and
weaknesses for making targeted transition mutations. When a single
target nucleotide is present within the base editing window, or when
bystander edits are acceptable, current base editors are typically more
efficient and generate fewer indels than prime editors. When multiple
cytosines or adenines are present and bystander edits are undesirable,
or when PAMs that position target nucleotides for base editing are not
available, prime editors offer substantial advantages.
Off-target prime editing
Prime editing requires target DNA–pegRNA spacer complementarity
for the Cas9 domain to bind, target DNA–pegRNA PBS complemen-
tarity to initiate pegRNA-templated reverse transcription, and target
DNA–RT product complementarity for flap resolution. To test whether
these three distinct DNA hybridization steps reduce off-target prime
editing compared to editing methods that require only target–guide
RNA complementarity, we treated HEK293T cells with PE3 or PE2 and
16 pegRNAs that target four genomic loci, each of which has at least
four well-characterized Cas9 off-target sites32,33. We also treated cells
with Cas9 nuclease and the same 16 pegRNAs, or with Cas9 and four
sgRNAs targeting the same four protospacers (Supplementary Table 1).
Consistent with previous studies32, Cas9 and sgRNAs targeting HEK3,
HEK4, EMX1, and FANCF modified the top four known Cas9 off-target
loci for each sgRNA with average frequencies of 16 ± 16%, 60 ± 26%,
48 ± 28%, and 4.3 ± 5.6%, respectively (Extended Data Fig. 6g). Cas9
with pegRNAs modified on-target sites with similar efficiency as Cas9
with sgRNAs, whereas Cas9 with pegRNAs modified off-target sites at
4.4-fold lower average efficiency than Cas9 with sgRNAs.
Strikingly, PE3 or PE2 with the same 16 pegRNAs containing these
four target spacers resulted in detectable off-target editing at only 3
out of 16 off-target sites, with only 1 of 16 showing an off-target editing
efficiency of 1% or more (Extended Data Fig. 6h). Average off-target
prime editing for pegRNAs targeting HEK3, HEK4, EMX1, and FANCF
at the top four known Cas9 off-target sites for each protospacer was
 a b c d g
Installation and correction of the Installation and correction of the Installation of the Insertion of a His6 tag, FLAG tag,
pathogenic E6V mutation pathogenic1278insTATC mutation protective G127V mutation PE3, DNMT1, +5 G to T, and extended LoxP sequence
in HBB in HEK293T cells in HEXA in HEK293T cells in PRNP in HEK293T cells mouse primary cortical neurons in HEK3 in HEK293T cells
Correct edit (without indels) Correct edit (without indels) Correct edit Correct edit Correct edit
80
Indels Indels (without indels) (without indels) (without indels)
40

the specified edit or indels (%)

the specified edit or indels (%)

Total sequencing reads with
Indels

the specified edit or indels (%)

Total sequencing reads with

Indels Indels
the specified edit or indels (%)

Total sequencing reads with

70 30
Total sequencing reads with

50

the specified edit or indels (%)

Total sequencing reads with
60 70 20 60
40 10
50 60
10
40 30 50
8 40
30 40
20 6
20 30
4
10 20 20
10 2
0 0 10
0
WT HBB E6V E6V HBB WT HEXA HEXA HEXA

Untreated
PE3
unsorted
PE3
sorted
Cas9
unsorted
Cas9
sorted
0 0
to E6V HBB to WT to +TATC +TATC +TATC to PRNP G127V His6 FLAG LoxP
(install) to WT with silent (install) to WT WT with silent (install)
(correct) (correct) (correct) (correct)
e PE3 in HEK293T, K562, U2OS, and HeLa cells f Cas9-initiated HDR in HEK293T, K562, U2OS, and HeLa cells
100 100

the specified edit or indels (%)

the specified edit or indels (%)

Total sequencing reads with

Total sequencing reads with

Correct edit (without indels)

Indels
80 80
Correct edit (without indels)
Indels
60 60

40 40

20 20

0 0
HEK3
+1 T to G
HEK3
+1 CTT ins
RNF2
+1 C to G
PRNP
+6 G to T

HEK3
+1 T to G
HEK3
+1 CTT ins
RNF2
+1 C to G

HEK3
+1 T to G
HEK3
+1 CTT ins
RNF2
+1 C to G

HEK3
+1 T to G
HEK3
+1 CTT ins
RNF2
+1 C to G

HEK3
+1 T to G
HEK3
+1 CTT ins
RNF2
+1 C to G
HBB E6V
(install)
HBB E6V
(correct)

PRNP
+6 G to T

HEK3
+1 T to G
HEK3
+1 CTT ins
RNF2
+1 C to G

HEK3
+1 T to G
HEK3
+1 CTT ins
RNF2
+1 C to G

HEK3
+1 T to G
HEK3
+1 CTT ins
RNF2
+1 C to G
HBB E6V
(install)
HBB E6V
(correct)
HEK293T cells K562 cells U2OS cells HeLa cells HEK293T cells K562 cells U2OS cells HeLa cells

Fig. 5 | Prime editing of pathogenic mutations, prime editing in primary Installation of a G•C-to-T•A transversion in DNMT1 of mouse primary cortical
mouse cortical neurons, and comparison of prime editing and HDR in four neurons using a split-intein PE3 lentivirus system (see Methods). Sorted values
human cell lines. a, Installation (via T•A-to-A•T transversion) and correction reflect editing or indels from GFP-positive nuclei, while unsorted values are
(via A•T-to-T•A transversion) of the pathogenic E6V-coding mutation in HBB in from all nuclei. e, f, PE3 editing and indels (e) or Cas9-initiated HDR editing and
HEK293T cells. Correction either to wild-type HBB, or to HBB containing a PAM- indels (f) at endogenous genomic loci in HEK293T, K562, U2OS, and HeLa cells.
disrupting silent mutation, is shown. b, Installation (via 4-bp insertion) and g, Targeted insertion of a His6 tag (18 bp), Flag epitope tag (24 bp), or extended
correction (via 4-bp deletion) of the pathogenic HEXA 1278+TATC allele in HEK293T loxP site (44 bp) in HEK293T cells by PE3. Editing efficiencies reflect
cells. Correction either to wild-type HEXA, or to HEXA containing a PAM- sequencing reads that contain the intended edit and do not contain indels
disrupting silent mutation, is shown. c, Installation of the protective G127V- among all treated cells, with no sorting, except where specified in e. Mean ± s.d.
coding variant in PRNP in HEK293T cells via G•C-to-T•A transversion. d, of n = 3 independent biological replicates.

<0.1%, <2.2 ± 5.2%, <0.1%, and <0.13 ± 0.11%, respectively (Extended Data PE2 minimally perturbed the transcriptome relative to Cas9 nickase or
Fig. 6h). Notably, at the HEK4 off-target 3 site that was edited by Cas9 a control lacking active RT (Supplementary Discussion).
with pegRNA1 at 97% efficiency, PE2 with pegRNA1 resulted in only 0.2%
off-target editing despite sharing the same pegRNA, demonstrating
how the two additional hybridization events required for prime edit- Prime editing pathogenic mutations
ing can greatly reduce off-target modification. Together, these results We tested the ability of PE3 to directly install or correct in human cells
suggest that prime editing induces much lower off-target editing than transversion, insertion, and deletion mutations that cause genetic
Cas9 at known Cas9 off-target sites. diseases. Sickle cell disease is caused by a A•T-to-T•A transversion muta-
Reverse transcription of 3′-extended pegRNAs in principle can tion in HBB, resulting in an E6V mutation in β-globin (Supplementary
proceed into the guide RNA scaffold, resulting in scaffold sequence Discussion). We used PE3 to install this HBB mutation into HEK293T
insertion that contributes to indels at the target locus. We analysed cells with 44% efficiency and 4.8% indels (Fig. 5a) and isolated from
66 PE3 editing experiments at four loci in HEK293T cells and observed a single prime editing experiment six HEK293T cell lines that were
1.7 ± 1.5% average total insertion of any number of pegRNA scaffold homozygous (triploid) for the mutated HBB allele (Supplementary
nucleotides (Extended Data Fig. 7). We speculate that inaccessibility Note 1). To correct the mutant HBB allele to wild-type HBB, we treated
of the guide RNA scaffold to reverse transcription due to Cas9 domain HEK293T cells homozygous for mutant HBB with PE3 and a pegRNA
binding, and cellular excision of the mismatched 3′ end of 3′ flaps that programmed to directly revert the HBB mutation to wild-type HBB.
extend into the pegRNA scaffold, minimize products that incorporate All 14 tested pegRNAs mediated efficient correction of mutant HBB to
pegRNA scaffold nucleotides. wild-type HBB (26–52% efficiency), and indel levels averaged 2.8 ± 0.70%
The presence of endogenous human RTs from retroelements34 and (Extended Data Fig. 9a). Introduction of a PAM-modifying silent muta-
telomerase suggests that RT activity is not inherently toxic to human tion improved editing efficiency and product purity to 58% correction
cells. Indeed, we observed no differences in the viability of HEK293T with 1.4% indels (Fig. 5a).
cells expressing dCas9, Cas9(H840A) nickase, PE2, or PE2 with R110S The most common mutation that causes Tay-Sachs disease is a 4-bp
and K103L mutations (PE2-dRT) that inactivate the RT and abolish insertion in HEXA (HEXA1278+TATC). We used PE3 to install this 4-bp inser-
prime editing35 (Extended Data Fig. 8a, b). To evaluate changes in the tion into HEXA with 31% efficiency and 0.8% indels (Fig. 5b), and iso-
cellular transcriptome that result from prime editing, we performed lated two HEK293T cell lines that were homozygous for HEXA1278+TATC
RNA sequencing (RNA-seq) on HEK293T cells expressing PE2, PE2- (Supplementary Note 1). We used these cells to test 43 pegRNAs and
dRT, or Cas9(H840A) nickase together with a PRNP-targeting or HEXA- three nicking sgRNAs with PE3 or PE3b systems for correction of the
targeting pegRNA (Extended Data Fig. 8c–k), and observed that active pathogenic insertion in HEXA (Extended Data Fig. 9b). Nineteen of the

Nature | www.nature.com | 7
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43 pegRNAs tested resulted in editing with an efficiency of 20% or more.
Correction to wild-type HEXA with the best pegRNA proceeded with 33%
efficiency and 0.32% indels using PE3b (Fig. 5b, Extended Data Fig. 9b).
Finally, we used PE3 to install a protective G•C-to-T•A transversion
into PRNP (resulting in PRNP(G127V)) into HEK293T cells, introducing
a mutant allele that confers resistance to prion disease in humans36 and
mice37 (Supplementary Discussion). We evaluated four pegRNAs and
three nicking sgRNAs. The most effective pegRNA with PE3 resulted
in 53% installation of G127V, with 1.7% indels (Fig. 5c). Together, these
results establish the ability of prime editing in human cells to install
or correct transversion, insertion, or deletion mutations that cause
or confer resistance to disease efficiently, and with few byproducts.
Other cell lines and primary neurons
Next, we tested prime editing at endogenous sites in three additional
human cell lines (Extended Data Fig. 10a, Supplementary Discussion).
In K562 cells, PE3 achieved three transversion edits and a His6 tag inser-
tion with 15–30% editing efficiency and 0.85–2.2% indels (Extended
Data Fig. 10a). In U2OS cells, we installed transversion mutations, as
well as a 3-bp insertion and His6 tag insertion, with 7.9–22% editing
efficiency and 0.13–2.2% indels (Extended Data Fig. 10a). Finally, in HeLa
cells we performed a 3-bp insertion with 12% average efficiency and
1.3% indels (Extended Data Fig. 10a). Collectively, these data indicate
that cell lines other than HEK293T support prime editing, although
editing efficiencies vary by cell type and are generally less efficient
than in HEK293T cells. Editing:indel ratios remained favourable in all
human cell lines tested.
To determine whether prime editing is possible in post-mitotic, ter-
minally differentiated primary cells, we transduced primary cortical
neurons from E18.5 mice with a PE3 lentiviral delivery system in which
PE2 protein components were expressed from the neuron-specific
synapsin promoter38 along with a GFP marker (see Methods). Nuclei
were isolated two weeks after transduction and sequenced directly,
or sorted for GFP expression before sequencing. We observed 7.1%
average prime editing of DNMT1 with 0.58% average indels in sorted
cortical neuron nuclei (Fig. 5d). Cas9 nuclease in the same lentivirus
system resulted in 31% average indels among sorted nuclei (Fig. 5d).
These data indicate that post-mitotic, terminally differentiated primary
cells can support prime editing.
Prime editing compared with HDR
Finally, we compared the performance of PE3 with that of optimized
Cas9-initiated HDR11,14 in mitotic cell lines that support HDR14. We
treated HEK293T, HeLa, K562 and U2OS cells with Cas9 nuclease, an
sgRNA, and a single-stranded DNA (ssDNA) donor template designed to
install a variety of transversion and insertion edits (Fig. 5e, f, Extended
Data Fig. 10). Cas9-initiated HDR in all cases successfully installed the
desired edit, but with far higher levels of indel byproducts than with PE3,
as expected given that Cas9 induces DSBs. In HEK293T cells, the ratio
of editing to indels for installation or correction of the allele encoding
HBB(E6V) or installation of the allele encoding PRNP(G127V) was on
average 270-fold higher for PE3 than for Cas9-initiated HDR.
Comparisons between PE3 and HDR in human cell lines other than
HEK293T showed similar results, although with lower PE3 editing effi-
ciencies (Fig. 5e, f, Supplementary Discussion). Collectively, these data
indicate that HDR typically results in similar or lower editing efficiencies
than PE3 with far more indels in four tested human cell lines (Extended
Data Fig. 10).
Discussion and future directions
The ability to insert arbitrary DNA sequences with single-nucleotide
precision is an especially promising capability of prime editing. For
8 | Nature | www.nature.com
example, we used PE3 in HEK293T cells to precisely insert into HEK3 a
His6 tag (18 bp, 65% efficiency), a Flag epitope tag (24 bp, 18% efficiency),
and an extended Cre recombinase loxP site (44 bp, 23% efficiency) with
3.0–5.9% indels (Fig. 5g). We anticipate that the ability to efficiently and
precisely insert new DNA sequences into target sites in living cells will
enable many biotechnological and therapeutic applications.
Collectively, the prime editing experiments described here performed
19 insertions up to 44 bp, 23 deletions up to 80 bp, 119 point mutations
including 83 transversions, and 18 combination edits at 12 endogenous
loci in the human and mouse genomes at locations ranging from 3 bp
upstream to 29 bp downstream of a PAM without making explicit DSBs.
These results establish prime editing as a remarkably versatile genome
editing method. Because 85–99% of insertions, deletions, indels, and
duplications in ClinVar are 30 bp in length or smaller (Extended Data
Fig. 11), in principle prime editing could correct up to about 89% of the
75,122 pathogenic human genetic variants in ClinVar (Fig. 1a).
Prime editing offers many possible choices of pegRNA-induced
nick locations, sgRNA-induced second nick locations, PBS lengths, RT
template lengths, and which strand to edit first. This flexibility, which
contrasts with more limited options typically available for other preci-
sion editing methods11,15,16, allows editing efficiency, product purity,
DNA specificity, and other parameters to be optimized to suit a given
application (Extended Data Fig. 9).
Much additional research is needed to further understand and
improve prime editing in a broad range of cell types and organisms,
to assess off-target prime editing in a genome-wide manner, and to
further characterize the extent to which prime editors might affect
cells. Interfacing prime editing with additional in vitro and in vivo
delivery strategies is essential for exploring the potential of prime
editing to enable applications, including the study and treatment of
genetic diseases. By enabling precise targeted transitions, transver-
sions, insertions, and deletions in the genomes of mammalian cells
without requiring DSBs, donor DNA templates, or HDR, however, prime
editing provides a new search-and-replace capability that substantially
expands the scope of genome editing.
Online content
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maries, source data, extended data, supplementary information,
acknowledgements, peer review information; details of author con-
tributions and competing interests; and statements of data and code
availability are available at https://doi.org/10.1038/s41586-019-1711-4.
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Article
Methods
General methods
DNA amplification was conducted by PCR using Phusion U Green Mul-
tiplex PCR Master Mix (ThermoFisher Scientific) or Q5 Hot Start High-
Fidelity 2× Master Mix (New England BioLabs) unless otherwise noted.
DNA oligonucleotides, including Cy5-labelled DNA oligonucleotides,
dCas9 protein, and Cas9(H840A) protein were obtained from Integrated
DNA Technologies. Yeast reporter plasmids were derived from previ-
ously described plasmids39 and cloned by the Gibson assembly method.
All mammalian editor plasmids used in this work were assembled using
the USER cloning method as previously described40. Plasmids expressing
sgRNAs were constructed by ligation of annealed oligonucleotides into
BsmBI-digested acceptor vector (Addgene plasmid no. 65777). Plasmids
expressing pegRNAs were constructed by Gibson assembly or Golden
Gate assembly using a custom acceptor plasmid (see Supplementary
Note 3). Sequences of sgRNA and pegRNA constructs used in this work
are listed in Supplementary Tables 2 and 3. All vectors for mammalian
cell experiments were purified using Plasmid Plus Midiprep kits (Qiagen)
or PureYield plasmid miniprep kits (Promega), which include endotoxin
removal steps. All experiments using live animals were approved by
the Broad Institute Institutional and Animal Care and Use Committees.
Wild-type C57BL/6 mice were obtained from Charles River (#027). No
statistical methods were used to predetermine sample size. The experi-
ments were not randomized, and investigators were not blinded to
allocation during experiments and outcome assessment.
In vitro biochemical assays
pegRNAs and sgRNAs were transcribed in vitro using the HiScribe T7
in vitro transcription kit (New England Biolabs) from PCR-amplified
templates containing a T7 promoter sequence. RNA was purified by
denaturing urea PAGE and quality-confirmed by an analytical gel before
use. 5′-Cy5-labelled DNA duplex substrates were annealed using two
oligonucleotides (Cy5-AVA024 and AVA025; 1:1.1 ratio) for the non-
nicked substrate or three oligonucleotides (Cy5-AVA023, AVA025 and
AVA026; 1:1.1:1.1) for the pre-nicked substrate by heating to 95 °C for
3 min followed by slowly cooling to room temperature (Supplementary
Table 2). Cas9 cleavage and reverse transcription reactions were carried
out in 1× cleavage buffer41 supplemented with dNTPs (20 mM HEPES-K,
pH 7.5; 100 mM KCl; 5% glycerol; 0.2 mM EDTA, pH 8.0; 3 mM MgCl2;
0.5 mM dNTP mix; 5 mM DTT). dCas9 or Cas9(H840A) (5 µM final) and
the sgRNA or pegRNA (5 µM final) were pre-incubated at room tempera-
ture in a 5-µl reaction mixture for 10 min before the addition of 0.5 μl of
4 μM duplex DNA substrate (400 nM final), followed by the addition of
0.2 μl of Superscript III reverse transcriptase (ThermoFisher Scientific),
an undisclosed M-MLV RT variant, when applicable. Reactions were
carried out at 37 °C for 1 h, then diluted to a volume of 10 µl with water,
treated with 0.2 µl of proteinase K solution (20 mg/ml, ThermoFisher
Scientific), and incubated at room temperature for 30 min. Following
heat inactivation at 95 °C for 10 min, reaction products were combined
with 2× formamide gel loading buffer (90% formamide; 10% glycerol;
0.01% bromophenol blue), denatured at 95 °C for 5 min, and separated
by denaturing urea PAGE gel (15% TBE-urea, 55 °C, 200 V). DNA products
were visualized by Cy5 fluorescence signal using a Typhoon FLA 7000
biomolecular imager.
Electrophoretic mobility shift assays were carried out in 1× binding
buffer (1× cleavage buffer with 10 µg/ml heparin) using pre-incubated
dCas9–sgRNA or dCas9–pegRNA complexes (concentration between
5 nM and 1 µM final) and Cy5-labelled duplex DNA (Cy5-AVA024 and
AVA025; 20 nM final). After 15 min of incubation at 37 °C, the samples were
analysed by native PAGE gel (10% TBE) and imaged for Cy5 fluorescence.
For DNA sequencing of reverse transcription products, fluorescent
bands were excised and purified from urea PAGE gels, then 3′ tailed
with terminal transferase (TdT; New England Biolabs) in the presence
of dGTP or dATP according to the manufacturer’s protocol. Tailed DNA
products were diluted tenfold with binding buffer (40% saturated
aqueous guanidinium chloride and 60% isopropanol) and purified
by QIAquick spin column (Qiagen), then used as templates for primer
extension by Klenow fragment (New England Biolabs) using primer
AVA134 (A-tailed products) or AVA135 (G-tailed products) (Supple-
mentary Table 2). Extensions were amplified by PCR for 10 cycles using
primers AVA110 and AVA122, then sequenced with AVA037 using the
Sanger method (Supplementary Table 2).
Yeast fluorescent reporter assays
Dual fluorescent reporter plasmids containing an in-frame stop codon,
a +1 frameshift, or a −1 frameshift were subjected to 5′-extended pegRNA
or 3′-extended pegRNA prime editing reactions in vitro as described
above using 100 ng of plasmid substrate. Following incubation at 37 °C
for 1 h, the reactions were diluted with water and plasmid DNA was
precipitated with 0.3 M sodium acetate and 70% ethanol. Resuspended
DNA was transformed into Saccharomyces cerevisiae by electroporation
as previously described42 and plated on synthetic complete medium
without leucine (SC(glucose), L−). GFP and mCherry fluorescence
signals were visualized from colonies with the Typhoon FLA 7000
biomolecular imager.
General mammalian cell culture conditions
HEK293T (ATCC CRL-3216), U2OS (ATTC HTB-96), K562 (CCL-243), and
HeLa (CCL-2) cells were purchased from ATCC and cultured and pas-
saged in Dulbecco’s modified Eagle’s medium (DMEM) plus GlutaMAX
(ThermoFisher Scientific), McCoy’s 5A medium (Gibco), RPMI medium
1640 plus GlutaMAX (Gibco), or Eagle’s minimal essential medium
(EMEM, ATCC), respectively, each supplemented with 10% (v/v) fetal
bovine serum (Gibco, qualified) and 1× penicillin streptomycin (Corn-
ing). All cell types were incubated, maintained, and cultured at 37 °C
with 5% CO2. Cell lines were authenticated by their respective suppliers
and tested negative for mycoplasma.
HEK293T tissue culture transfection protocol and genomic DNA
preparation
HEK293T cells were seeded on 48-well poly-d-lysine coated plates
(Corning). Between 16 and 24 h after seeding, cells were transfected
at approximately 60% confluency with 1 µl lipofectamine 2000 (Thermo
Fisher Scientific) according to the manufacturer’s protocols and 750 ng
PE plasmid, 250 ng pegRNA plasmid, and 83 ng sgRNA plasmid (for
PE3 and PE3b). Unless otherwise stated, cells were cultured for 3 days
following transfection, after which the medium was removed, the
cells were washed with 1× PBS solution (Thermo Fisher Scientific),
and genomic DNA was extracted by the addition of 150 µl of freshly
prepared lysis buffer (10 mM Tris-HCl, pH 7.5; 0.05% SDS; 25 µg/ml
proteinase K (ThermoFisher Scientific)) directly into each well of the
tissue culture plate. The genomic DNA mixture was incubated at 37 °C
for 1–2 h, followed by an 80 °C enzyme inactivation step for 30 min.
Primers used for mammalian cell genomic DNA amplification are listed
in Supplementary Table 4. For HDR experiments in HEK293T cells,
231 ng Cas9 nuclease-expression plasmid, 69 ng sgRNA-expression
plasmid and 50 ng (1.51 pmol) of 100-nt ssDNA donor template (PAGE-
purified; Integrated DNA Technologies) was lipofected using 1.4 µl
lipofectamine 2000 (ThermoFisher) per well. Genomic DNA from all
HDR experiments was purified using the Agencourt DNAdvance Kit
(Beckman Coulter), according to the manufacturer’s protocol.
High-throughput DNA sequencing of genomic DNA samples
Genomic sites of interest were amplified from genomic DNA samples
and sequenced on an Illumina MiSeq as previously described with the
following modifications17,18. In brief, amplification primers containing
Illumina forward and reverse adapters (Supplementary Table 4) were
used for a first round of PCR (PCR 1) to amplify the genomic region of
interest. PCR 1 reactions (25 µl) were performed with 0.5 µM of each
forward and reverse primer, 1 µl genomic DNA extract and 12.5 µl Phu-
sion U Green Multiplex PCR Master Mix. PCR reactions were carried out
as follows: 98 °C for 2 min, then 30 cycles of [98 °C for 10 s, 61 °C for
20 s, and 72 °C for 30 s], followed by a final 72 °C extension for 2 min.
Unique Illumina barcoding primer pairs were added to each sample in
a secondary PCR reaction (PCR 2). Specifically, 25 µl of a given PCR 2
reaction contained 0.5 µM of each unique forward and reverse Illumina
barcoding primer pair, 1 µl unpurified PCR 1 reaction mixture, and 12.5 µl
Phusion U Green Multiplex PCR 2× Master Mix. The barcoding PCR 2
reactions were carried out as follows: 98 °C for 2 min, then 12 cycles
of [98 °C for 10 s, 61 °C for 20 s, and 72 °C for 30 s], followed by a final
72 °C extension for 2 min. PCR products were evaluated analytically by
electrophoresis in a 1.5% agarose gel. PCR 2 products (pooled by com-
mon amplicons) were purified by electrophoresis with a 1.5% agarose
gel using a QIAquick Gel Extraction Kit (Qiagen), eluting with 40 µl
water. DNA concentration was measured by fluorometric quantification
(Qubit, ThermoFisher Scientific) or qPCR (KAPA Library Quantification
Kit-Illumina, KAPA Biosystems) and sequenced on an Illumina MiSeq
instrument according to the manufacturer’s protocols.
Sequencing reads were demultiplexed using MiSeq Reporter (Illu-
mina). Alignment of amplicon sequences to a reference sequence was
performed using CRISPResso243. For all prime editing yield quantifica-
tion, prime editing efficiency was calculated as: percentage of (number
of reads with the desired edit that do not contain indels)/(number of
total reads). For quantification of point mutation editing, CRISPResso2
was run in standard mode with “discard_indel_reads” on. Prime edit-
ing for installation of point mutations was then explicitly calculated
as: (frequency of specified point mutation in non-discarded reads) ×
(number of non-discarded reads)/(total reads). For insertion or dele-
tion edits, CRISPResso2 was run in HDR mode using the desired allele
as the expected allele (e flag), and with “discard_indel_reads” on. Editing
yield was calculated as: (number of HDR-aligned reads)/(total reads).
For all experiments, indel yields were calculated as: (number of indel-
containing reads)/(total reads).
Nucleofection of U2OS, K562, and HeLa cells
Nucleofection was used for transfection in all experiments using K562,
HeLa, and U2OS cells. For PE conditions in these cell types, 800 ng
prime editor expression plasmid, 200 ng pegRNA expression plasmid,
and 83 ng nicking sgRNA expression plasmid was nucleofected in a
final volume of 20 μl in a 16-well nucleocuvette strip (Lonza). For HDR
conditions in these three cell types, 350 ng Cas9 nuclease expression
plasmid, 150 ng sgRNA expression plasmid and 200 pmol (6.6 μg) 100-
nt ssDNA donor template (PAGE-purified; Integrated DNA Technolo-
gies) was nucleofected in a final volume of 20 µl per sample in a 16-well
Nucleocuvette strip (Lonza). K562 cells were nucleofected using the
SF Cell Line 4D-Nucleofector X Kit (Lonza) with 5 × 105 cells per sample
(program FF-120), according to the manufacturer’s protocol. U2OS
cells were nucleofected using the SE Cell Line 4D-Nucleofector X Kit
(Lonza) with 3–4 × 105 cells per sample (program DN-100), according
to the manufacturer’s protocol. HeLa cells were nucleofected using the
SE Cell Line 4D-Nucleofector X Kit (Lonza) with 2 × 105 cells per sample
(program CN-114), according to the manufacturer’s protocol. Cells
were harvested 72 h after nucleofection for genomic DNA extraction.
Genomic DNA extraction for HDR experiments
Genomic DNA from all HDR comparison experiments in HEK293T,
HEK293T HBB(E6V), K562, U2OS, and HeLa cells was purified using
the Agencourt DNAdvance Kit (Beckman Coulter), according to the
manufacturer’s protocol.
Comparison between PE2, PE3, BE2, BE4max, ABEdmax, and
ABEmax
HEK293T cells were seeded on 48-well poly-d-lysine coated plates
(Corning). After 16–24 h, cells were transfected at approximately 60%
confluency. For base editing with CBE or ABE constructs, cells were
transfected with 750 ng base editor plasmid, 250 ng sgRNA expression
plasmid, and 1 µl of lipofectamine 2000 (Thermo Fisher Scientific).
PE transfections were performed as described above. Genomic DNA
extraction for PE and BE was performed as described above.
Determination of PE3 activity at known Cas9 off-target sites
To evaluate PE3 off-target editing activity at known Cas9 off-target sites,
genomic DNA extracted from HEK293T cells 3 days after transfection
with PE3 was used as template for PCR amplification of 16 previously
reported Cas9 off-target genomic sites32,33 (the top four off-target sites
each for the HEK3, EMX1, FANCF, and HEK4 spacers; primer sequences
are listed in Supplementary Table 4). These genomic DNA samples were
identical to those used for quantifying on-target PE3 editing activi-
ties shown in Fig. 4 or Extended Data Fig. 5d, e; pegRNA and nicking
sgRNA sequences are listed in Supplementary Table 3. Following PCR
amplification of off-target sites, amplicons were sequenced on the
Illumina MiSeq platform as described above (see ‘High-throughput
DNA sequencing of genomic DNA samples’ section). To determine the
on-target and off-target editing activity of Cas9 nuclease, Cas9(H840A)
nickase, dCas9, and PE2-dRT, we transfected HEK293T cells with 750 ng
editor plasmid (Cas9 nuclease, Cas9(H840A) nickase, dCas9, or PE2-
dRT), 250 ng pegRNA or sgRNA plasmid, and 1 μl lipofectamine 2000.
Genomic DNA was isolated from cells 3 days after transfection as
described above. On-target and off-target genomic loci were ampli-
fied by PCR using the primer sequences in Supplementary Table 4 and
sequenced on an Illumina MiSeq.
High-throughput sequencing (HTS) data analysis was performed
using CRISPResso243. The editing efficiencies of Cas9 nuclease, Cas9
H840A nickase, and dCas9 were quantified as the percentage of total
sequencing reads containing indels. For quantification of PE3 and
PE3-dRT off-targets, aligned sequencing reads were examined for
point mutations, insertions, or deletions that were consistent with
the anticipated product of pegRNA reverse transcription initiated at
the Cas9 nick site. Single nucleotide variations occurring at <0.1% over-
all frequency among total reads within a sample were excluded from
analysis. For reads containing single nucleotide variations that both
occurred at frequencies ≥0.1% and were partially consistent with the
pegRNA-encoded edit, t-tests (unpaired, one-tailed, α = 0.5) were used
to determine whether the variants occurred at significantly higher lev-
els compared to samples treated with pegRNAs that contained the same
spacer but encoded different edits. To avoid differences in sequencing
errors, comparisons were made between samples that were sequenced
simultaneously within the same MiSeq run. Variants that did not meet
the criteria of P > 0.05 were excluded. Off-target PE3 editing activity
was then calculated as the percentage of total sequencing reads that
met the above criteria.
Generation of a HEK293T cell line containing HBB(E6V) using
Cas9-initiated HDR
HEK293T cells were seeded in a 48-well plate and transfected at
approximately 60% confluency with 1.5 µl lipofectamine 2000, 300 ng
Cas9(D10A) nickase plasmid, 100 ng sgRNA plasmid, and 200 ng
100-mer ssDNA donor template (Supplementary Table 5). Three days
after transfection, the medium was exchanged for fresh medium. Four
days after transfection, cells were dissociated using 30 µl TrypLE solu-
tion and suspended in 1.5 ml medium. Single cells were isolated into
individual wells of two 96-well plates by fluorescence-activated cell
sorting (FACS) (Beckman-Coulter Astrios). See Supplementary Note
1 for representative FACS sorting examples. Cells were expanded for
14 days before genomic DNA sequencing as described above. Of the
isolated clonal populations, none was found to be homozygous for the
HBB allele encoding the E6V mutation, so a second round of editing by
lipofection, sorting, and outgrowth was repeated in a partially edited
cell line to yield a cell line homozygous for the E6V-encoding allele.
Article
Generation of a HEK293T cell line containing HBB(E6V) using PE3
HEK293T cells (2.5 × 104) were seeded on 48-well poly-d-lysine coated
plates (Corning). Between 16 and 24 h after seeding, cells were trans-
fected at approximately 70% confluency with 1 µl lipofectamine 2000
(Thermo Fisher Scientific) according to the manufacturer’s protocols
and 750 ng PE2-P2A-GFP plasmid, 250 ng pegRNA plasmid, and 83 ng
sgRNA plasmid. After 3 days, cells were washed with 1× PBS (Gibco) and
dissociated using TrypLE Express (Gibco). Cells were then diluted with
DMEM plus GlutaMax (Thermo Fisher Scientific) supplemented with
10% (v/v) FBS (Gibco) and passed through a 35-µm cell strainer (Corn-
ing) before sorting. Flow cytometry was carried out on a LE-MA900 cell
sorter (Sony). Cells were treated with 3 nM DAPI (BioLegend) 15 min
before sorting. After gating for doublet exclusion, single DAPI-negative
cells with GFP fluorescence above that of a GFP-negative control cell
population were sorted into 96-well flat-bottom cell culture plates
(Corning) filled with pre-chilled DMEM with GlutaMax supplemented
with 10% FBS. See Supplementary Note 1 for representative FACS sort-
ing examples and allele tables. Cells were cultured for 10 days before
genomic DNA extraction and characterization by HTS, as described
above. A total of six clonal cell lines were identified that are homozygous
for the E6V-encoding mutation in HBB.
Generation of a HEK293T cell line containing the HEXA1278+TATC
insertion using PE3
HEK293T cells containing the HEXA1278+TATC allele were generated fol-
lowing the protocol described above for creation of the HBB(E6V) cell
line; pegRNA and sgRNA sequences are listed in Supplementary Table 3
under the Fig. 5 subheading. After transfection and sorting, cells were
cultured for 10 days before genomic DNA was extracted and character-
ized by HTS, as described above. We recovered two heterozygous cell
lines that contained 50% HEXA1278+TATC alleles and two homozygous cell
lines containing 100% HEXA1278+TATC alleles.
Cell viability assays
HEK293T cells were seeded in 48-well plates and transfected at approxi-
mately 70% confluency with 750 ng editor plasmid (PE2, PE2(R110S/
K103L), Cas9(H840A) nickase, or dCas9), 250 ng HEK3-targeting
pegRNA plasmid, and 1 μl lipofectamine 2000, as described above.
Cell viability was measured every 24 h post-transfection for 3 days using
the CellTiter-Glo 2.0 assay (Promega) according to the manufacturer’s
protocol. Luminescence was measured in 96-well flat-bottomed poly-
styrene microplates (Corning) using a M1000 Pro microplate reader
(Tecan) with a 1-s integration time.
Lentivirus production
Lentivirus was produced as previously described44. T-75 flasks of rapidly
dividing HEK293T cells (ATCC; Manassas, VA, USA) were transfected
with lentivirus production helper plasmids pVSV-G and psPAX2 in com-
bination with modified lentiCRISPRv2 genomes carrying intein-split
PE2 editor using FuGENE HD (Promega, Madison, WI, USA) according
to the manufacturer's protocol. Four split-intein editor constructs
were designed: 1) a viral genome encoding a U6-pegRNA expression
cassette and the N-terminal portion (1–573) of Cas9(H840A) nickase
fused to the Npu N-intein, a self-cleaving P2A peptide, and GFP-KASH;
2) a viral genome encoding the Npu C-intein fused to the C-terminal
remainder of PE2; 3) a viral genome encoding the Npu C-intein fused
to the C-terminal remainder of Cas9 for the Cas9 control; and 4) a nick-
ing sgRNA for DNMT1 (derived from Addgene plasmid no. 52963).  The
split-intein45 mediates trans splicing to join the two halves of PE2 or
Cas9, while the P2A GFP-KASH enables co-translational production of a
nuclear membrane-localized GFP. After 48 h, supernatant was collected,
centrifuged at 500g for 5 min to remove cellular debris, and filtered
using a 0.45-µm filter. Filtered supernatant was concentrated using the
PEG-it Virus Precipitation Solution (System Biosciences, Palo Alto, CA,
USA) according to the manufacturer’s directions. The resulting pellet
was resuspended in Opti-MEM (Thermo Fisher Scientific, Waltham,
MA, USA) using 1% of the original medium volume. Resuspended pellet
was flash-frozen and stored at −80 °C until use.
Mouse primary cortical neuron dissection and culture
E18.5 dissociated cortical cultures were taken from timed-pregnant
C57BL/6 mice (Charles River). Embryos were removed from pregnant
mice after euthanasia by CO2 followed by decapitation. Cortical caps
were dissected in ice-cold Hibernate-E supplemented with penicil-
lin/streptomycin (Life Technologies). Following a rinse with ice-cold
Hibernate-E, tissue was digested at 37 °C for 8 min in papain/DNase
(Worthington/Sigma). Tissue was triturated in NBActiv4 (BrainBits) sup-
plemented with DNase. Cells were counted and plated in 24-well plates at
100,000 cells per well. Half of the medium was changed twice per week.
Prime editing in primary neurons and nucleus isolation
At days in vitro (DIV) 1, 15 µl lentivirus was added at a 10:10:1 ratio of
N-terminal:C-terminal:nicking sgRNA. At DIV 14, neuronal nuclei were
isolated using the EZ-PREP buffer (Sigma D8938) following the manu-
facturer’s protocol. All steps were performed on ice or at 4 °C. Medium
was removed from dissociated cultures, and cultures were washed
with ice-cold PBS. PBS was aspirated and replaced with 200 µl EZ-PREP
solution. Following a 5-min incubation on ice, EZ-PREP was pipetted
across the surface of the well to dislodge remaining cells. The sam-
ple was centrifuged at 500g for 5 min, and the supernatant removed.
Samples were washed with 200 µl EZ-PREP and centrifuged again at
500g for 5 min. Samples were resuspended with gentle pipetting in
200 µl ice-cold Nuclei Suspension Buffer (NSB) consisting of 100 µg/
ml BSA and 3.33 µM Vybrant DyeCycle Ruby (Thermo Fisher) in 1×PBS,
then centrifuged at 500g for 5 min. The supernatant was removed and
nuclei were resuspended in 100 µl NSB and sorted into 100 µl Agencourt
DNAdvance lysis buffer using a MoFlo Astrios (Beckman Coulter) at
the Broad Institute flow cytometry facility. Genomic DNA was purified
according to the manufacturer’s Agencourt DNAdvance instructions.
RNA-seq and data analysis
HEK293T cells were co-transfected with PRNP-targeting or HEXA-target-
ing pegRNAs and PE2, PE2-dRT, or Cas9(H840A) nickase. Seventy-two
hours after transfection, total RNA was harvested from cells using TRI-
zol reagent (Thermo Fisher) and purified with RNeasy Mini kit (Qiagen)
including on-column DNaseI treatment. Ribosomes were depleted from
total RNA using the rRNA removal protocol of the TruSeq Stranded
Total RNA library prep kit (Illumina) and subsequently washed with
RNAClean XP beads (Beckman Coulter). Sequencing libraries were
prepared using ribo-depleted RNA on a SMARTer PrepX Apollo NGS
library prep system (Takara) following the manufacturer’s protocol.
The resulting libraries were visualized on a 2200 TapeStation (Agilent
Technologies), normalized using a Qubit dsDNA HS assay (Thermo
Fisher), and sequenced on a NextSeq 550 using high output v2 flow
cell (Illumina) as 75-bp paired-end reads. Fastq files were generated
with bcl2fastq2 version 2.20 and trimmed using TrimGalore version
0.6.2 (https://github.com/FelixKrueger/TrimGalore) to remove low-
quality bases, unpaired sequences, and adaptor sequences. Trimmed
reads were aligned to a Homo sapiens genome assembly GRCh38 with a
custom Cas9(H840A) gene entry using RSEM version 1.3.146. The limma-
voom47 package was used to normalize gene expression levels and
perform differential expression analysis with batch effect correction.
Differentially expressed genes were called with FDR-corrected P < 0.05
and fold change > 2 cutoffs, and results were visualized in R.
ClinVar analysis
The ClinVar variant summary was downloaded from NCBI (accessed July
15, 2019), and the information contained therein was used for all down-
stream analysis. The list of all reported variants was filtered by allele
ID in order to remove duplicates and by clinical significance in order
to restrict the analysis to pathogenic variants. The list of pathogenic
variants was filtered sequentially by variant type in order to calculate
the fraction of pathogenic variants that are insertions, deletions, and
so on. Single nucleotide variants (SNVs) were separated into two cat-
egories (transitions and transversions) on the basis of the reported
reference and alternate alleles. SNVs that did not report reference or
alternate alleles were excluded from the analysis.
The lengths of reported insertions, deletions, and duplications were
calculated using reference/alternate alleles, variant start/stop posi-
tions, or appropriate identifying information in the variant name. Vari-
ants that did not report any of the above information were excluded
from the analysis. The lengths of reported indels (single variants that
include both insertions and deletions relative to the reference genome)
were calculated by determining the number of mismatches or gaps
in the best pairwise alignment between the reference and alternate
alleles. Frequency distributions of variant lengths were calculated
using GraphPad Prism 8.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.
Data availability
High-throughput sequencing data have been deposited to the NCBI
Sequence Read Archive database under accession PRJNA565979.
Plasmids encoding PE1, PE2 (same as PE3), and pegRNA expression
vectors are available from Addgene. Previously described plasmids
expressing sgRNAs are also available from Addgene, such as Addgene
plasmid no. 65777.
Code availability
The script used to quantify pegRNA scaffold insertion is provided
as Supplementary Note 4.
39. Anzalone, A. V., Lin, A. J., Zairis, S., Rabadan, R. & Cornish, V. W. Reprogramming
eukaryotic translation with ligand-responsive synthetic RNA switches. Nat. Methods 13,
453–458 (2016).
40. Badran, A. H. et al. Continuous evolution of Bacillus thuringiensis toxins overcomes insect
resistance. Nature 533, 58–63 (2016).
41. Anders, C. & Jinek, M. in Methods in Enzymology (eds. Doudna, J. A. & Sontheimer, E. J.)
546, 1–20 (Academic, 2014).
42. Pirakitikulr, N., Ostrov, N., Peralta-Yahya, P. & Cornish, V. W. PCRless library mutagenesis
via oligonucleotide recombination in yeast. Protein Sci. 19, 2336–2346 (2010).
43. Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence
analysis. Nat. Biotechnol. 37, 224–226 (2019).
44. Levy, J. M. & Nicoll, R. A. Membrane-associated guanylate kinase dynamics reveal regional
and developmental specificity of synapse stability. J. Physiol. (Lond.) 595, 1699–1709 (2017).
45. Zettler, J., Schütz, V. & Mootz, H. D. The naturally split Npu DnaE intein exhibits an
extraordinarily high rate in the protein trans-splicing reaction. FEBS Lett. 583, 909–914
(2009).
46. Li, B. & Dewey, C. N. RSEM: accurate transcript quantification from RNA-seq data with or
without a reference genome. BMC Bioinformatics 12, 323 (2011).
47. Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing
and microarray studies. Nucleic Acids Res. 43, e47 (2015).
Acknowledgements We thank J. M. Madison for neuron cell culture advice. This work was
supported by the Merkin Institute of Transformative Technologies in Healthcare, US NIH grants
U01AI142756, RM1HG009490, R01EB022376, and R35GM118062, and the HHMI. A.V.A.
acknowledges a Jane Coffin Childs postdoctoral fellowship. P.B.R. and A.R. acknowledge NIH
T32 GM095450. A.A.S. acknowledges NIH T32 GM007726. P.J.C. and A.R. acknowledge NSF
graduate fellowships. C.W. acknowledges a Damon Runyon Cancer Research Foundation
fellowship (DRG-2343-18). G.A.N acknowledges a Helen Hay Whitney postdoctoral fellowship.
Author contributions A.V.A. designed the research, performed experiments, analysed data,
and wrote the manuscript. P.B.R., J.R.D., A.A.S., and G.A.N. performed human cell experiments
and analysed data. L.W.K. and J.M.L. performed neuron experiments. P.J.C. and C.W. performed
and analysed RNA-seq experiments. A.R. analysed ClinVar data. D.R.L designed and supervised
the research and wrote the manuscript.
Competing interests Authors through the Broad Institute have filed patent applications on
prime editing. D.R.L. is a consultant and co-founder of Prime Medicine, Beam Therapeutics,
Pairwise Plants, and Editas Medicine, companies that use genome editing.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41586-019-
1711-4.
Correspondence and requests for materials should be addressed to D.R.L.
Peer review information Nature thanks Guangping Gao, Randall Platt and Fyodor Urnov for
their contribution to the peer review of this work.
Reprints and permissions information is available at http://www.nature.com/reprints.
Article
a b c
HEK3
Electrophoretic mobility shift assays 100
HEK293T cells

% of total sequencing reads

5’-extended pegRNAs
1.0 dsDNA substrate 80
sgRNA
Fraction of DNA bound

pegRNA 1

with indels
60
pegRNA 2
pegRNA 3
40
pegRNA 4
0.5
pegRNA 5
20
Nicked products
0

3’-extended

5’-extended
sgRNA

pegRNA

pegRNA
dsDNA substrate + + +
0.0 Cas9 H840A nickase – + +
1 10 100 1000
5’-extended pegRNA – + –
[dCas9:gRNA] (nM) 3’-extended pegRNA – – +
d
Pre-nicked Cy5-labeled DNA substrate
Cy5 sgRNA or Reverse 5’
Protospacer PAM 5’-extended pegRNA or 3’-extended transcripta
transcriptase
3’-extended pegRNA pegRNA 3’
5’ 3’ + 3’
dCas9 or Cas9 H840A Cy5
3’ 5’ Cy5
5
5’ 5’
5
OR OR 5’
5’
M-MLV RT 3’ 3’
Non-nicked Cy5-labeled DNA substrate dNTPs
Cy5 37 ˚C, 1 hr
Protospacer PAM Separate primer extension 5’-extended
products by denaturing Cas9 pegRNA
5’ 3’ urea PAGE and visualize
3’ 5’ Cy5 fluorescence

e 5’-extended pegRNAs f 5’-extended pegRNAs

Non-nicked
DNA substrate RT
RT products products

Pre-nicked
DNA substrate Cleavage
products

Nicked ssDNA marker + – – – – – – – Nicked ssDNA marker – + – – – – – –

Pre-nicked DNA – + + + + + + + dsDNA substrate + – + + + + + +
dCas9 – – + + + + + + Cas9 H840A nickase – – + + + + + +
M-MLV RT – – + + + + + + M-MLV RT – – – + + + + +
sgRNA – – + – – – – – sgRNA – – + – – – – –
5’-extended pegRNA – – – + + + + + 5’-extended pegRNA – – – + + + + +
RT template length (nt) – – – 7 7 8 15 22 RT template length (nt) – – – 7 7 8 15 22
Linker – – – A B B B B Linker – – – A B B B B

g gRNA
RT template scaffold homopolymer tail
3’-extended pegRNA

RT product
poly-G tailing

Band excision,
DNA elution,
TdT tailing,
primer extension,
Sanger sequencing poly-A tailing

Extended Data Fig. 1 | See next page for caption.

Extended Data Fig. 1 | In vitro prime editing validation studies with
fluorescently labelled DNA substrates. a, Electrophoretic mobility shift
assays with dCas9, 5′-extended pegRNAs and 5′-Cy5-labelled DNA substrates.
pegRNAs 1–5 contain a 15-nt linker sequence (linker A for pegRNA 1, linker B for
pegRNAs 2–5) between the spacer and the PBS, a 5-nt PBS sequence, and RT
templates of 7 nt (pegRNAs 1 and 2), 8 nt (pegRNA 3), 15 nt (pegRNA 4), and 22 nt
(pegRNA 5). pegRNAs are those used in e and f; full sequences are listed
in Supplementary Table 2. b, In vitro nicking assays of Cas9(H840A) using
5′-extended and 3′-extended pegRNAs. Data in a, b are representative of n = 2
independent replicates. c, Cas9-mediated indel formation in HEK293T cells at
HEK3 using 5′-extended and 3′-extended pegRNAs. Mean ± s.d. of n = 3
independent biological replicates. d, Overview of prime editing in vitro
biochemical assays. 5′-Cy5-labelled pre-nicked and non-nicked dsDNA
substrates were tested. sgRNAs, 5′-extended pegRNAs, or 3′-extended
pegRNAs were pre-complexed with dCas9 or Cas9(H840A) nickase, then
combined with dsDNA substrate, Superscript III M-MLV RT, and dNTPs.
Reactions were allowed to proceed at 37 °C for 1 h before separation by
denaturing urea PAGE and visualization by Cy5 fluorescence. e, Primer
extension reactions using 5′-extended pegRNAs, pre-nicked DNA substrates,
and dCas9 lead to substantial conversion to RT products. f, Primer extension
reactions using 5′-extended pegRNAs as in b with non-nicked DNA substrate
and Cas9(H840A) nickase. Product yields are greatly reduced by comparison
to pre-nicked substrate. g, An in vitro primer extension reaction using a
3′-pegRNA generates a single apparent product by denaturing urea PAGE. The
RT product band was excised, eluted from the gel, then subjected to
homopolymer tailing with terminal transferase (TdT) using either dGTP or
dATP. Tailed products were extended using poly-T or poly-C primers, and the
resulting DNA was sequenced. Sanger traces indicate that three nucleotides
derived from the pegRNA scaffold were reverse-transcribed (added as the final
3′ nucleotides to the DNA product). Note that pegRNA scaffold insertion is
much rarer in mammalian cell prime editing experiments than in vitro
(Extended Data Fig. 6), potentially owing to the inability of the tethered RT to
access the Cas9-bound guide RNA scaffold, and/or cellular excision of
mismatched 3′ ends of 3′ flaps containing pegRNA scaffold sequences. Data in
e–g are representative of n = 2 independent replicates. For gel source data,
see Supplementary Fig. 1.
Article
a b
Stop codon,
+1 frameshift, or 1. Cas9 H840A, No Unedited
-1 frameshift GFP only
pegRNA, dNTPs, editing GFP-STOP-mCherry
M-MLV RT negative control

GFP mCherry 2. Transform

into yeast cells Editing
GFP–mCherry Pre-edited
fusion protein GFP–mCherry
positive control
Start
c d
3’-Extended pegRNA stop codon correction (T•A to A•T) 5’-Extended pegRNA stop codon correction (T•A to A•T)

mCherry GFP merge mCherry GFP merge

e f
3’-Extended pegRNA +1 frameshift correction (1-bp deletion) 3’-Extended pegRNA -1 frameshift correction (1-bp insertion)

mCherry GFP merge mCherry GFP merge

g
Sequence of additional GFP and
pegRNA-encoded T-to-A edit
mCherry double-positive colonies

Unedited plasmid
transformant
(GFP-only colony)

Edited plasmid
transformant
(GFP and mCherry
double-positive
colony)
Extended Data Fig. 2 | Cellular repair in yeast of 3′ DNA flaps from in vitro
prime editing reactions. a, Dual fluorescent protein reporter plasmids
contain GFP and mCherry open reading frames separated by a target site
encoding an in-frame stop codon, a +1 frameshift, or a −1 frameshift. Prime
editing reactions were carried out in vitro with Cas9(H840A) nickase, pegRNA,
dNTPs, and M-MLV RT, then transformed into yeast. Colonies that contain
unedited plasmids produce GFP but not mCherry. Yeast colonies containing
edited plasmids produce both GFP and mCherry as a fusion protein. b, Overlay
of GFP and mCherry fluorescence for yeast colonies transformed with reporter
plasmids containing a stop codon between GFP and mCherry (unedited
negative control, top), or containing no stop codon or frameshift between GFP
and mCherry (pre-edited positive control, bottom). c–f, Visualization of
mCherry and GFP fluorescence from yeast colonies transformed with in vitro
prime editing reaction products. c, d, Stop codon correction via T•A-to-A•T
transversion using a 3′-extended pegRNA (c) or a 5′-extended pegRNA (d). e, +1
frameshift correction via a 1-bp deletion using a 3′-extended pegRNA. f, −1
frameshift correction via a 1-bp insertion using a 3′-extended pegRNA.
g, Sanger DNA sequencing traces from plasmids isolated from GFP-only
colonies in b and GFP and mCherry double-positive colonies in c. Data in b–g
are representative of n = 2 independent replicates.
Extended Data Fig. 3 | Prime editing of genomic DNA in human cells by PE1.
a, pegRNAs contain a spacer sequence, an sgRNA scaffold, and a 3′ extension
containing an RT template (purple), which contains the edited base(s) (red),
and a primer-binding site (PBS, green). The primer-binding site hybridizes to
the nicked target DNA strand. The RT template is homologous to the DNA
sequence downstream of the nick, with the exception of the encoded edited
base(s). b, Installation of a T•A-to-A•T transversion at the HEK3 site in HEK293T
cells using Cas9(H840A) nickase fused to wild-type M-MLV RT (PE1) and
pegRNAs with varying PBS lengths. c, T•A-to-A•T transversion editing
efficiency and indel generation by PE1 at the +1 position of HEK3 using pegRNAs
containing 10-nt RT templates and PBS sequences ranging from 8 to 17 nt.
d, G•C-to-T•A transversion editing efficiency and indel generation by PE1 at the
+5 position of EMX1 using pegRNAs containing 13-nt RT templates and PBS
sequences ranging from 9 to 17 nt. e, G•C-to-T•A transversion editing efficiency
and indel generation by PE1 at the +5 position of FANCF using pegRNAs
containing 17-nt RT templates and PBS sequences ranging from 8 to 17 nt.
f, C•G-to-A•T transversion editing efficiency and indel generation by PE1 at the
+1 position of RNF2 using pegRNAs containing 11-nt RT templates and PBS
sequences ranging from 9 to 17 nt. g, G•C-to-T•A transversion editing efficiency
and indel generation by PE1 at the +2 position of HEK4 using pegRNAs
containing 13-nt RT templates and PBS sequences ranging from 7 to 15 nt.
h, PE1-mediated +1 T deletion, +1 A insertion, and +1 CTT insertion at the HEK3
site using a 13-nt PBS and a 10-nt RT template. Sequences of pegRNAs are as in
Fig. 2a (Supplementary Table 3). Editing efficiencies reflect sequencing reads
that contain the intended edit and do not contain indels among all treated cells,
with no sorting. Mean ± s.d. of n = 3 independent biological replicates.
 i
f
c
a

o
% of total sequencing reads with % of total sequencing reads with
% of total sequencing reads with target G•C converted to C•G target G•C converted to C•G
% of total sequencing reads
target G•C converted to C•G

0
5
10
15
0
5
10
15
20
with the specified edit or indels
PE1

PE2

0
5
10
15
20

0
2
4
6
8
10
untreated untreated PE1-M1

PE1-M3
PE1-M2

PE1-M6
Untreated

PE1-M15
PE1 PE1

PE1-M3inv
PE1-M3 PE1-M1 PE1-M1
Article

PE1-M3+T306K PE1-M2 PE1-M2

Indels
PE1-M3+ PE1-M3 PE1-M3
E302R+W313F

HEK3 +2 G to C
PE2 PE1-M3+E607K PE1-M3+E607K
PE1-M3+ PE1-M3+
E607K+L139P E607K+L139P
PE1-M6 PE1-M6

PE2, HEK3 +1 T to A

PBS length (nucleotides)

PE1-M3+L139P

j
PE1-M3+L139P

Correct edit
(w/o indels)
% of total sequencing reads with PE1-M3+E69K PE1-M3+E69K

8 9 10 11 12 13 14 15 16 17
targeted FLAG insertion

0
2
4
6
PE1-M3+T306K PE1-M3+T306K

p
Untreated PE1-M3+E302R PE1-M3+E302R
HEK3 +2 G to C

EMX1 +1 G to C
Cas9 H840A–M-MLV RT (wt)

% of total sequencing reads

with the specified edit or indels PE1-M3+W313F PE1-M3+W313F
PE1-M3
PE1-M3+ PE1-M3+
Cas9 H840A–M-MLV RT (D200N)

5
10
15
PE1-M3+T306K E302R+W313F E302R+W313F
PE1-M3+ PE1-M3+L435G PE1-M3+L435G
E302R+W313F PE1-M3+N454K PE1-M3+N454K
PE2
Cas9 H840A–M-MLV RT (D200N+L603W)

PE1-M15 PE1-M15

Indels
HEK3 FLAG-tag insertion (24 bp)
PE1-M15+D200N PE1-M15+D200N

Correct edit
(w/o indels)
PE1-M3inv PE1-M3inv
M-MLV RT (D200N+L603W+T330P)–Cas9 H840A
Cas9 H840A–M-MLV RT (D200N+L603W+T330P)

g
d

% of total sequencing reads with % of total sequencing reads with

k
target T•A converted to A•T targeted FLAG insertion

PE2, EMX1 +5 G to T
% of total sequencing reads with
0
2
4
6

0
2
4
6
8
10
target C•G converted to A•T

PBS length (nucleotides)

0
2
4
6
8
untreated
untreated

9 10 11 12 13 14 15 16 17
Untreated PE1
PE1

q
Cas9 H840A–M-MLV RT (D200N+L603W+T330P+T306K+W313F)

% of total sequencing reads PE1-M3 PE1-M1 PE1-M1

with the specified edit or indels
PE1-M3+T306K PE1-M2 PE1-M2

Abbreviations for prime editor variants used in this figure. b, Targeted

0
5
10
15
20
PE1-M3+ PE1-M3
PE1-M3
Cas9 H840A–M-MLV RT (D200N+L603W+T330P+D524G+E562Q+D583N)

E302R+W313F

RNF2 +1 C to A
PE1-M3+E607K

and a +1 G•C-to-C•G transversion edit at FANCF (h). i–n, Comparison of four

PE2 PE1-M3+E607K

a +2 G•C-to-C•G transversion edit at HEK3 (c), a 24-bp Flag insertion at the +1

PE1-M3+

C•G transversion edit at EMX1 (f), a +2 T•A-to-A•T transversion edit at HBB (g),
position of HEK3 (d), a +1 C•G-to-A•T transversion edit at RNF2 (e), a +1 G•C-to-
PE1-M3+

insertion and deletion edits with PE1 at the HEK3 locus. c–h, Comparison of 18
E607K+L139P

Extended Data Fig. 4 | Evaluation of M-MLV RT variants for prime editing. a,

E607K+L139P

prime editor constructs containing M-MLV RT variants for their ability to install
PE1-M6
PE1-M6
PE1-M3+L139P
PE1-M3+L139P

Indels
PE1-M3+E69K
% of total sequencing reads with PE1-M3+E69K
target G•C converted to C•G PE1-M3+T306K

Correct edit
(w/o indels)
PE1-M3+T306K

PE2, FANCF +5 G to T
0
5
10
15
PE1-M3+E302R
HBB +2 T to A

PBS length (nucleotides)

PE1-M3+E302R
Untreated PE1-M3+W313F
PE1-M3+W313F
PE1-M3+

8 9 10 11 12 13 14 15 16 17
PE1-M3 PE1-M3+
E302R+W313F

r
HEK3 FLAG-tag insertion (24 bp)

E302R+W313F
PE1-M3+T306K PE1-M3+L435G
% of total sequencing reads PE1-M3+L435G
PE1-M3+ PE1-M3+N454K
with the specified edit or indels PE1-M3+N454K
E302R+W313F EMX1 +1 G to C

0
1
2
3
4
PE2 PE1-M15
PE1-M15
PE1-M15+D200N
PE1-M15+D200N
PE1-M3inv
PE1-M3inv

Indels
e

% of total sequencing reads with % of total sequencing reads with

target G•C converted to C•G target C•G converted to A•T

Correct edit
m

(w/o indels)
% of total sequencing reads with
0
2
4
6
0
1
2
3
4

target T•A converted to A•T

2
4
6
8

untreated untreated

PE2, RNF2 +1 C to A
Untreated PE1 PE1

PBS length (nucleotides)

PE1-M3 PE1-M1 PE1-M1

PE1-M3+T306K PE1-M2 PE1-M2

9 10 11 12 13 14 15 16 17
PE1-M3+ PE1-M3 PE1-M3
HBB +2 T to A

E302R+W313F
Cas9 H840A–M-MLV RT (P51L+S67R+E67K+T197A+H204R+E302K+F309N+W313F+T330P+L435G+N454K+D524G+D583N+H594Q+D653N)

s
PE2 PE1-M3+E607K PE1-M3+E607K
% of total sequencing reads PE1-M3+ PE1-M3+
b

with the specified edit or indels E607K+L139P E607K+L139P

% of total sequencing reads
PE1-M6 PE1-M6

0
2
4
6
8
10
with the specified edit or indels
0
5
10
15
20

PE1-M3+L139P PE1-M3+L139P

7
PE1-M3+E69K PE1-M3+E69K
n

% of total sequencing reads with +1 T del

target G•C converted to C•G PE1-M3+T306K PE1-M3+T306K
0
1
2
3
4
5

PE1-M3+E302R PE1-M3+E302R
RNF2 +1 C to A

+1 A ins
FANCF +1 G to C

Untreated PE1-M3+W313F PE1-M3+W313F

PE1, HEK3

PE1-M3 PE1-M3+ PE1-M3+

E302R+W313F E302R+W313F +1 CTT ins
PE2, HEK4 +2 G to T

PE1-M3+T306K PE1-M3+L435G PE1-M3+L435G

Indels

PE1-M3+ PE1-M3+N454K PE1-M3+N454K

with no sorting. Mean ± s.d. of n = 3 independent biological replicates.

E302R+W313F
Correct edit
(w/o indels)

PBS length (nucleotides)

FANCF +1 G to C

PE2 PE1-M15 PE1-M15

8 9 10 11 12 13 14 15
Indels

PE1-M15+D200N PE1-M15+D200N

RNF2. s, +2 G•C-to-T•A at HEK4. Editing efficiencies reflect sequencing reads

prime editor constructs containing M-MLV variants for their ability to install
PE1-M3inv PE1-M3inv
Correct edit

at HEK3. p, +5 G•C-to-T•A at EMX1. q, +5 G•C-to-T•A at FANCF. r, +1 C•G-to-A•T at

(w/o indels)

the edits shown in c–h in a second round of independent experiments. o–s, PE2
editing efficiency at five genomic loci with varying PBS lengths. o, +1 T•A-to-A•T

that contain the intended edit and do not contain indels among all treated cells,
 a
40 PE2, 13-nt PBS, VEGFA +5 G to T
with the specified edit or indels Correct edit
% of total sequencing reads

(w/o indels)
30 Indels

20

10

0
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
RT template length (nucleotides)
Last templated
nucleotide T C C A G A T G G C A C A T T G T C A G A G G G

b
15 PE2, 13-nt PBS, DNMT1 +5 G to T
with the specified edit or indels

Correct edit
% of total sequencing reads

(w/o indels)
Indels
10

0
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
RT template length (nucleotides)
Last templated
nucleotide T G A C G G G A G G G C A G A A C T A G T C C T

c
PE2, 15-nt PBS, RUNX1 +5 G to T
with the specified edit or indels

25
Correct edit
% of total sequencing reads

(w/o indels)
20
Indels
15

10

0
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
RT template length (nucleotides)
Last templated T T C A G A C A G C A T A T T T G A G T C A T T
nucleotide

d e f
PE3, 17-nt RT template, PE3, 16-nt template, PE3, 11-nt RT template,
nicking at +48, FANCF nicking at +53, EMX1 nicking at +49, DNMT1
60 40 50
Correct edit
with the specified edit or indels
with the specified edit or indels
with the specified edit or indels

(w/o indels) Correct edit Correct edit

% of total sequencing reads
% of total sequencing reads
% of total sequencing reads

50 (w/o indels) (w/o indels)

Indels 40
30 Indels Indels
40
30
30 20
20
20
10
10
10

0 0 0
+4 G to C

+9 C to G
+3 A to G

+5 G to A
+5 G to T

+8 T to G
+2 A to C

+7 C to A

+8 T to C

+9 C to T
+2 A to T

+8 T to A

+2 C to G

+6 G to C
+4 G to A
+5 G to T

+8 T to G
+10 G to T

+1 A to G
+2 C to A

+2 C to T

+8 T to C
+1 A to C

+8 T to A
+3 A to T
+3 C to G

+6 G to C
+1 A to G

+4 T to G
+5 G to A
+2 C to A

+3 C to T

+8 T to C
+7 A to C
+1 A to T

+4 T to A

Extended Data Fig. 5 | Design features of pegRNA PBS and RT template pegRNA) are highlighted in red; RT templates that end in C should be avoided
sequences, and additional editing examples with PE3. a, PE2-mediated +5 during pegRNA design to maximize prime editing efficiency. b, +5 G•C-to-T•A
G•C-to-T•A transversion editing efficiency (blue line) at VEGFA in HEK293T cells transversion editing and indels for DNMT1 as in a. c, +5 G•C-to-T•A transversion
as a function of RT template length. Indels (grey line) are plotted for editing and indels for RUNX1 as in a. d–f, PE3-mediated transition and
comparison. The sequence below the graph shows the last nucleotide transversion edits at the specified positions for FANCF (d), EMX1 (e), and DNMT1
templated for synthesis by the pegRNA. G nucleotides (templated by a C in the (f). Mean ± s.d. of n = 3 independent biological replicates.
Article
a b
HEK3 total C•G-to-T•A conversion FANCF tota C•G-to-T•A conversion EMX1 total C•G-to-T•A conversion Indels
% of total sequencing reads with

% of total sequencing reads with

% of total sequencing reads with
80 80 80 BE2max BE4max PE2 80
BE2max BE4max
target C•G converted to T•A

target C•G converted to T•A

target C•G converted to T•A
BE2max PE2 PE3

% of total sequencing
PE2 PE3 PE3
PE2 BE2max BE4max

reads with indels

60 60 60 60
BE4max
40 40 PE3 40 40

20 20 20 20

0 0 0 0

CBE

CBE

CBE
C3
C4
C7

C3
C7
C8

C5
C6
C7
C3 C4 C7 C3 C7 C8 C5 C6 C7

HEK3 FANCF EMX1

c
HEK3 precise C•G-to-T•A edits FANCF precise C•G-to-T•A edits EMX1 precise C•G-to-T•A edits

on y the target C•G converted to T•A

only the target C•G converted to T•A

BE2max

% of total sequencing reads with

% of total sequencing reads with

BE2max BE4max 60 30

base pair(s) converted to A•T

60 BE2max

% of total sequencing reads

with on y the specified C•G
PE2
PE2 PE3 PE2 BE4max
40 40 BE4max 20 PE3
PE3

20 20 10

0 0 0
C3 C4 C7 C3 C7 C8 C5 C6 C7 C5+C6 C5+C7 C6+C7 C5+C6+C7
d e f
HEK3 tota A•T-to-G•C conversion FANCF tota A•T-to-G•C conversion HEK3 precise A•T-to-G•C edits FANCF precise A•T-to-G•C edits Indels

only the target A•T converted to G•C

only the target A•T converted to G•C

% of total sequencing reads with
ABEdmax ABEmax
% of total sequencing reads with

% of total sequencing reads with

% of total sequencing reads with

80 50 ABEdmax 80 ABEdmax ABEmax 50 ABEdmax 80 ABEdmax
target A•T converted to G•C

target A•T converted to G•C

PE2 PE3 PE2 PE2 PE3 PE2 PE2

% of total sequencing
40 40
60 60 ABEmax 60 ABEmax

reads with indels

ABEmax
30 PE3 30 PE3 PE3
40 40 40
20 20
20 20 20
10 10

0 0 0 0 0

ABE

ABE
A5
A8

A5
A5 A8 A5 A5 A8 A5

HEK3 FANCF
g
Cas9-mediated % indels
HEK3 HEK4 EMX1 FANCF
pegRNA pegRNA pegRNA pegRNA
NA

NA

NA

NA
Guide RNA
sgR

sgR

sgR

sgR
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
100
On-target 91.8 87.5 89.2 89.1 86.8 71.8 68.6 72.8 72.8 70.9 85.6 79.7 70.6 76.6 76.0 78.7 55.9 58.3 51.8 52.0
80
Off-target site 1 17.2 1.9 5.5 5.2 1.8 54.2 39.5 48.4 49.7 49.2 81.1 63.5 48.1 53.0 59.6 12.6 1.9 1.9 1.7 1.7
60
Off-target site 2 38.0 6.5 12.6 11.8 4.7 42.5 19.5 29.4 27.3 30.3 58.3 12.0 6.0 8.2 12.9 1.1 0.2 0.2 0.2 0.1
40
Off-target site 3 8.8 0.6 1.7 1.5 0.5 98.1 96.9 97.3 97.6 97.5 14.8 4.2 3.1 3.6 4.8 2.4 0.2 0.1 0.2 0.2
20
Off-target site 4 0.3 0.1 0.1 0.1 < 0.1 45.3 16.9 28.0 27.5 29.7 39.5 1.3 0.9 0.6 1.3 1.0 0.2 0.2 0.2 0.2
0

h
PE-mediated % editing and % indels (in parentheses)
HEK3 (PE3) HEK4 (PE2) EMX1 (PE3) FANCF (PE3)
pegRNA pegRNA pegRNA pegRNA
Guide RNA
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
100
44.2 61.2 40.4 48.4 18.2 14.4 9.8 7.9 28.6 14.1 35.7 15.4 56.9 32.4 42.8 47.6
On-target (11.9) (8.8) (16.5) (3.3) (0.9) (1.8) (2.0) (2.2) (3.5) (2.4) (3.3) (2.9) (9.3) (16.7) (13.6) (12.0)
80
< 0.1 < 0.1 < 0.1 < 0.1 < 0.1 0.4 < 0.1 0.4 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 0.6 < 0.1 < 0.1
Off-target site 1 (< 0.1) (< 0.1) (< 0.1) (< 0.1) (< 0.1) (< 0.1) (< 0.1) (< 0.1) (0.1) (0.1) (0.1) (0.1) (0.1) (0.1) (0.1) (0.1)
60
< 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1
Off-target site 2 (< 0.1) (< 0.1) (< 0.1) (< 0.1) (0.1) (0.1) (0.1) (0.1) (< 0.1) (0.1) (0.1) (0.1) (< 0.1) (< 0.1) (0.1) (< 0.1)
40
< 0.1 < 0.1 < 0.1 < 0.1 0.2 6.8 19.2 7.9 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1
Off-target site 3 (< 0.1) (< 0.1) (< 0.1) (< 0.1) (0.5) (1.9) (0.5) (3.5) (0.3) (0.3) (0.3) (0.3) (< 0.1) (< 0.1) (< 0.1) (< 0.1)
20
< 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1
Off-target site 4 (0.1) (0.1) (0.1) (< 0.1) (< 0.1) (0.1) (0.2) (< 0.1) (0.1) (0.1) (0.2) (0.1) (< 0.1) (< 0.1) (< 0.1) (< 0.1)
0

Extended Data Fig. 6 | Comparison of prime editing and base editing, and HEK293T cells at four endogenous on-target sites and their 16 known top off-
off-target editing by Cas9 and prime editors at known Cas9 off-target sites. target sites32,33. For each on-target site, Cas9 was paired with an sgRNA or with
a, C•G-to-T•A editing efficiency at the same target nucleotides for PE2, PE3, each of four pegRNAs that recognize the same protospacer. h, Average
BE2max, and BE4max at endogenous HEK3, FANCF, and EMX1 sites in HEK293T triplicate on-target and off-target editing efficiencies and indel efficiencies
cells. b, Indel frequency from treatments in a. c, Editing efficiency of precise (below in parentheses) in HEK293T cells for PE2 or PE3 paired with each
C•G-to-T•A edits (without bystander edits or indels) at HEK3, FANCF, and EMX1. pegRNA in g. Editing efficiencies reflect sequencing reads that contain the
d, Total A•T-to-G•C editing efficiency for PE2, PE3, ABEdmax, and ABEmax at intended edit and do not contain indels among all treated cells, with no sorting.
HEK3 and FANCF. e, Precise A•T-to-G•C editing efficiency without bystander Off-target editing efficiencies in h reflect off-target locus modification
edits or indels at HEK3 and FANCF. f, Indel frequency from treatments in d. consistent with prime editing. Mean ± s.d. of n = 3 independent biological
g, Average triplicate Cas9 nuclease editing efficiencies (indel frequencies) in replicates.
 a +5 G to A +9 C to T +3 A to G +6 T ins
EMX1

Cumulative % of total sequencing reads

% of total sequencing reads with
scaffold insertion of any length 1.5 0.6 1.5 +4 G to C +8 T to A +2 A to T +1 TGC ins
+5 G to A +9 C to T

pegRNA / scaffold insertion of the

% of total sequencing reads with

with pegRNA scaffold insertion

+4 G to C +8 T to A +5 G to T +8 T to C +7 C to A +5 G del

exact length specified

+5 G to T +8 T to C +2 A to C +8 T to G +9 C to G +4-6 GGG del
1.0
0.4 1.0
+2 A to C +8 T to G
+3 A to G +6 T ins
0.5
+2 A to T +1 TGC ins
0.2 0.5
+7 C to A +5 G del
0.0 +9 C to G +4-6 GGG del
+1 TGC ins
+2 A to C
+2 A to T
+3 A to G
+4 G to C
+4-6 GGG del
+5 G del
+5 G to A
+5 G to T
+6 T ins
+7 C to A
+8 T to A
+8 T to C
+8 T to G
+9 C to G
+9 C to T
0.0 0.0
0 20 40 60 80 100 0 20 40 60 80 100
pegRNA scaffold insertion length (nt) pegRNA scaffold insertion length (nt)
pegRNA

b
+5 G to A +3 C to T +1 A to G +3 C ins
FANCF

Cumulative % of total sequencing reads

+5 G to A +3 C to T 8
% of total sequencing reads with

8
scaffold insertion of any length

pegRNA / scaffold insertion of the +6 G to C +4 T to A +1 A to T +4 GAT ins

% of total sequencing reads with

with pegRNA scaffold insertion

+6 G to C +4 T to A
1.0 +10 G to T +8 T to C +2 C to A +6 G del
6 +10 G to T +8 T to C
exact length specified
6
+7 A to C +4 T to G +3 C to G +5-7 GGA del
+7 A to C +4 T to G
4 +1 A to G +3 C ins
4
0.5 +1 A to T +4 GAT ins
2
+2 C to A +6 G del
2
+3 C to G +5-7 GGA del
0
+1 A to G
+1 A to T
+2 C to A
+3 C to G
+3 C to T

+8 T to C
+10 G to T
+3 C ins
+4 T to A
+4 T to G
+4 GAT ins
+5 G to A
+5-7 GGA del
+6 G to C
+6 G del
+7 A to C

0.0 0
0 20 40 60 80 100 0 20 40 60 80 100
pegRNA scaffold insertion length (nt) pegRNA scaffold insertion length (nt)
pegRNA

c +1 T to A +4 T to A
HEK3 +1 T to A +4 T to A +2 G to T +5 G to T

Cumulative % of total sequencing reads

+1 T to C +4 T to C
% of total sequencing reads with

1.0 0.6 1.5

scaffold insertion of any length

pegRNA / scaffold insertion of the

+1 T to C +4 T to C +6 G to A
% of total sequencing reads with

+3 A to G
+1 T to G

with pegRNA scaffold insertion

+4 T to G
0.8 +1 T to G +4 T to G +3 A to T +6 G to C
exact length specified

+2 G to A +5 G to A
+2 G to A +5 G to A +3 A to C +6 G to T
0.6 0.4 +2 G to C +5 G to C 1.0
+2 G to C +5 G to C
+2 G to T +5 G to T
0.4
+3 A to G +6 G to A
0.2 0.2 +3 A to T +6 G to C 0.5

+3 A to C +6 G to T
0.0
+1 T to A
+1 T to C
+1 T to G
+2 G to A
+2 G to C
+2 G to T
+3 A to C
+3 A to G
+3 A to T
+4 T to A
+4 T to C
+4 T to G
+5 G to A
+5 G to C
+5 G to T
+6 G to A
+6 G to C
+6 G to T

0.0 0.0
0 20 40 60 80 100 0 20 40 60 80 100
pegRNA pegRNA scaffold insertion length (nt) pegRNA scaffold insertion length (nt)

d +6 G to A +1 C to T +4 A to G +1 T ins
RNF2
Cumulative % of total sequencing reads
% of total sequencing reads with

1.5 8
pegRNA / scaffold insertion of the

6
scaffold insertion of any length

+6 G to A +1 C to T
% of total sequencing reads with

+3 G to C +2 T to A +4 A to T +1 GTA ins
with pegRNA scaffold insertion

+3 G to C +2 T to A +5 G to T +7 T to C +1 C to A +4 A del
exact length specified

+5 G to T +7 T to C 6
4 +4 A to C +2 T to G +1 C to G +3-5 GAG del
1.0
+4 A to C +2 T to G
+4 A to G +1 T ins 4
2
+4 A to T +1 GTA ins
0.5
+1 C to A +4 A del
2
0 +1 C to G +3-5 GAG del
+1 C to A
+1 C to G
+1 C to T
+1 T ins
+1 GTA ins
+2 T to A
+2 T to G
+3 G to C
+3-5 GAG del
+4 A to C
+4 A to G
+4 A to T
+4 A del
+5 G to T
+6 G to A
+7 T to C

0.0 0
0 20 40 60 80 100 0 20 40 60 80 100
pegRNA scaffold insertion length (nt) pegRNA scaffold insertion length (nt)
pegRNA

Extended Data Fig. 7 | Incorporation of pegRNA scaffold sequence into percentage of total sequencing reads containing a pegRNA scaffold sequence
target loci. HTS data were analysed for pegRNA scaffold sequence insertion as insertion of the specified length (middle); and the cumulative total percentage
described in Supplementary Note 4. a, Analysis for the EMX1 locus. Shown is the of pegRNA insertion up to and including the length specified on the x-axis. b, As
percentage of total sequencing reads containing one or more pegRNA scaffold in a for FANCF. c, As in a for HEK3. d, As in a for RNF2. Mean ± s.d. of n = 3
sequence nucleotides within an insertion adjacent to the RT template (left); the independent biological replicates.
Article
a b
HEK3, +5 G to A
106 15

with the specified edit or indels

8 PE2

% of total sequencing reads

PE2 R110S K103L (dRT) Correct edit (w/o indels)
Cell viability (RLU)

6 106 10 Indels
Cas9 H840A nickase
dCas9
4 106 5
Untransfected
2 106
0

PE2

PE2-dRT

dCas9

Untransfected
Cas9 H840A
nickase
0
1 2 3
Days post-transfection

c d e
pegRNA targeting PRNP pegRNA targeting PRNP pegRNA targeting PRNP
–log10 (adjusted p-value)

–log10 (adjusted p-value)

–log10 (adjusted p-value)

log2 (PE2 / PE2-dRT) log2 (PE2 / Cas9 H840A) log2 (PE2-dRT / Cas9 H840A)

f g h
pegRNA targeting HEXA pegRNA targeting HEXA pegRNA targeting HEXA
–log10 (adjusted p-value)

–log10 (adjusted p-value)

–log10 (adjusted p-value)

log2 (PE2 / PE2-dRT) log2 (PE2 / Cas9 H840A) log2 (PE2-dRT / Cas9 H840A)

i j k
PRNP HEXA PRNP HEXA PRNP HEXA

Upregulated 40 18 32 Upregulated 13 11 45
Upregulated 3 2

Downregulated 9 7 15 Downregulated 19 25 18 28 16 4
Downregulated

PE2 / PE2-dRT PE2 / Cas9 H840A PE2-dRT / Cas9 H840A

Extended Data Fig. 8 | See next page for caption.

Extended Data Fig. 8 | Effects of PE2, PE2-dRT, Cas9(H840A) nickase, and
dCas9 on cell viability and on transcriptome-wide RNA abundance.
HEK293T cells were transiently transfected with plasmids encoding PE2,
PE2(R110S/K103L), Cas9(H840A) nickase, or dCas9, together with a HEK3-
targeting pegRNA plasmid. Cell viability was measured for the bulk cellular
population every 24 h after transfection for 3 days using the CellTiter-Glo 2.0
assay (Promega). a, Viability, as measured by luminescence, at 1, 2, or 3 days
after transfection. Mean ± s.e.m. of n = 3 independent biological replicates,
each performed in technical triplicate. b, Percentage editing and indels for PE2,
PE2(R110S/K103L), Cas9(H840A) nickase, or dCas9, together with a HEK3-
targeting pegRNA plasmid that encodes a +5 G-to-A edit. Editing efficiencies
were measured on day 3 after transfection from cells treated alongside those
used for assaying viability in a. Mean ± s.d. of n = 3 independent biological
replicates. c–k, Analysis of cellular RNA, depleted for ribosomal RNA, isolated
from HEK293T cells expressing PE2, PE2-dRT, or Cas9(H840A) nickase and a
PRNP-targeting or HEXA-targeting pegRNA. RNAs corresponding to 14,410
genes and 14,368 genes were detected in PRNP and HEXA samples, respectively.
c–h, Volcano plot displaying the −log10 FDR-adjusted P value versus log 2-fold
change in transcript abundance for each RNA, comparing PE2 versus PE2-dRT
with PRNP-targeting pegRNA (c), PE2 versus Cas9(H840A) with PRNP-targeting
pegRNA (d), PE2-dRT versus Cas9(H840A) with PRNP-targeting pegRNA (e),
PE2 versus PE2-dRT with HEXA-targeting pegRNA(f), PE2 versus Cas9(H840A)
with HEXA-targeting pegRNA (g), PE2-dRT versus Cas9(H840A) with HEXA-
targeting pegRNA (h). Red dots indicate genes that show twofold or more
changes in relative abundance that are statistically significant (FDR-adjusted
P < 0.05). i–k, Venn diagrams of upregulated and downregulated transcripts
(twofold change or more) comparing PRNP and HEXA samples for PE2 versus
PE2-dRT (i), PE2 versus Cas9(H840A) ( j), and PE2-dRT versus Cas9(H840A) (k).
Values for each RNA-seq condition reflect the mean of n = 5 biological
replicates. Differential expression was assessed using a two-sided t-test with
empirical Bayesian variance estimation.
Article
a
Correction of HBB E6V in HEK293T cells with PE3
Correct edit
% of total sequencing reads with

60
non-pathogenic allele or indels

(w/o indels)
Indels

40

20

0
Untreated
HBB 3.5
HBB 3.7
HBB 5.2
HBB 5.3
HBB 5.4
HBB 5.5
HBB 5.6
HBB 5.7
HBB 5.8
HBB 5.9
HBB 5.10
HBB 5.11
HBB 5.12
HBB 5.13
pegRNA
b
60
Correction of HEXA 1278+TATC in HEK293T cells with PE3
% of total sequencing reads with

Correct edit
non-pathogenic allele or indels

(w/o indels)
Indels
40

20

0
HEXAs 1
HEXAs 2
HEXAs 3
HEXAs 4
HEXAs 5
HEXAs 6
HEXAs 7
HEXAs 8
HEXAs 9
HEXAs 10
HEXAs 11
HEXAs 12
HEXAs 13
HEXAs 14
HEXAs 15
HEXAs 16
HEXAs 17
HEXAs 18
HEXAs 19
HEXAs 20
HEXAs 21
HEXAs 22
HEXAs 23
HEXAs 24
HEXAs 25
HEXAs 26
HEXA 5
HEXA 6
HEXA 7
HEXA 8
HEXA 9
HEXA 10
HEXA 11
HEXA 12
HEXA 13
HEXA 14
HEXA 15
HEXA 16
HEXA 17
HEXA 18
HEXA 19
HEXA 20
HEXA 21
HEXA 11 b
HEXAs 24 b
HEXAs 26 b
untreated 1
untreated 2
pegRNA

Extended Data Fig. 9 | PE3-mediated correction of E6V-encoding HBB Those pegRNAs labelled HEXAs correct the pathogenic allele by a shifted 4-bp
mutation and HEXA1278+TATC by various pegRNAs. a, Screen of 14 pegRNAs for deletion that disrupts the PAM and leaves a silent mutation. Those pegRNAs
correction of the HBB E6V-encoding allele in HEK293T cells with PE3. All labelled HEXA correct the pathogenic allele back to wild-type. Entries ending in
pegRNAs evaluated convert the mutant HBB allele back to wild-type HBB b use an edit-specific nicking sgRNA in combination with the pegRNA (the PE3b
without the introduction of any silent PAM mutation. b, Screen of 41 pegRNAs system). Mean ± s.d. of n = 3 independent biological replicates.
for correction of the HEXA 1278+TATC allele in HEK293T cells with PE3 or PE3b.
 a b HEK293T cells
PE3, K562 cells PE3, U2OS cells PE3, HeLa cells 100 Correct edit (w/o indels)

with the specified edit or indels

Indels

% of total sequencing reads

Correct edit Correct edit Correct edit 80
(w/o indels) (w/o indels) (w/o indels)

with the specified edit or indels

40 Indels 25 15 Indels

with the specified edit or indels

with the specified edit or indels
Indels

% of total sequencing reads

60

% of total sequencing reads

% of total sequencing reads
20
30
10 40
15
20 20
10
5
10 0
5

non-targeting
PE3
Cas9 HDR

non-targeting
PE3
Cas9 HDR

non-targeting
PE3
Cas9 HDR

PE3
Cas9 HDR

PE3
Cas9 HDR

non-targeting
PE3
Cas9 HDR

PE3
Cas9 HDR

PE3
Cas9 HDR

non-targeting

Cas9 HDR
PE3

PE3
Cas9 HDR
0 0 0

HEK3
+5 G to T
HEK3
+1 ins 6xHis

HEK3
+1 CTT ins
FANCF
+5 G to T
HEK3
+2 G to C

EMX1
+1 G to C
FANCF
+5 G to T
HEK3
+1 ins 6xHis

HEK3
+1 CTT ins
HEK3 HEK3 HEK3 HEK3 HEK3 RNF2 RNF2 RNF2 HBB PRNP
+1 T to G +3 A to C +3 A to T +3 A to T, +1 CTT ins +1 C to A +1 C to G +1 GTAins +4 A to T +6 G to T
+5-6 GG to TT
c d
K562 cells
80 U2OS cells
100 Correct edit (w/o indels)
with the specified edit or indels

Correct edit (w/o indels)

% of total sequencing reads

with the specified edit or indels

% of total sequencing reads
Indels Indels
60 80

60
40
40
20
20

0
0
non-targeting
PE3

Cas9 HDR
non-targeting

PE3
Cas9 HDR
non-targeting
PE3
Cas9 HDR

Cas9 HDR

PE3

Cas9 HDR
non-targeting
PE3

PE3
Cas9 HDR
non-targeting
PE3

Cas9 HDR
non-targeting

non-targeting
PE3

Cas9 HDR
non-targeting

PE3
Cas9 HDR
non-targeting
PE3
Cas9 HDR
non-targeting
PE3

Cas9 HDR
PE3

Cas9 HDR
non-targeting
PE3
Cas9 HDR
non-targeting
PE3

Cas9 HDR
HEK3 HEK3 HEK3 RNF2 RNF2 RNF2 HBB HEK3 HEK3 HEK3 RNF2 RNF2 RNF2 HBB
+1 T to G +3 A to C +1 CTT ins +1 C to A +1 C to G +1 GTA ins +4 A to T +1 T to G +3 A to C +1 CTT ins +1 C to A +1 C to G +1 GTA ins +4 A to T
e f
HeLa cells 100 HDR ssDNA template carryover controls
100
Correct edit (w/o indels) Correct edit (w/o indels)

with the specified edit or indels

with the specified edit or indels
% of total sequencing reads

80

% of total sequencing reads

80 Indels Indels

60 60

40
40

20
20
0
non-targeting
PE3

Cas9 HDR
non-targeting

PE3
Cas9 HDR
non-targeting
PE3
Cas9 HDR
non-targeting
PE3

Cas9 HDR
PE3

Cas9 HDR
non-targeting
PE3
Cas9 HDR
non-targeting
PE3

Cas9 HDR

non-targeting

dCas9 HDR

wt Cas9 HDR
non-targeting

dCas9 HDR

wt Cas9 HDR

non-targeting

dCas9 HDR

wt Cas9 HDR

non-targeting

dCas9 HDR

wt Cas9 HDR

non-targeting

dCas9 HDR

wt Cas9 HDR
non-targeting

dCas9 HDR

wt Cas9 HDR
HEK3 HEK3 HEK3 RNF2 RNF2 RNF2 HBB
+1 T to G +3 A to C +1 CTT ins +1 C to A +1 C to G +1 GTA ins +4 A to T
K562, HEK3 U2OS, HEK3 HeLa, HEK3 HEK293T, HEK3 HEK293T, HBB K562, RNF2
g +3 A to T +3 A to T +3 A to T +3 A to T +4 T to A
(E6V correction)
+1 C to G

T G G G G C C C A G A C T G A G C A C G T G A T G G C A G A G G A A A G G A A G Reference
K562 HEK3 negative control (non-targeting pegRNA) Substitutions
T G G G G C C C A G A C T G A G C A C G T G A T G G C A G A G G A A A G G A A G
93.54% (47190 reads) Insertions
T G G G G C C C A G A C T G A G C A C G T G A T G G C A G A G G A A A G G A A G
0.47% (238 reads) Deletions
T G G G G C C C A G A C T G A G C A C G T G A T G G C A G C G G A A A G G A A G
0.21% (108 reads) Predicted
K562 HEK3 PE3 +1 CTT insertion cleavage position
T G G G G C C C A G A C T G A G C A C G T G A T G G C A G A G G A A A G G A A G
69.66% (39061 reads)
T G G G G C C C A G A C T G A G C A C G C T T T G A T G G C A G A G G A A A G G
22.80% (12784 reads)
T G G G G C C C A G A C T G A G C A C G T G A T G G C A G A G G A A A G G A A G
0.32% (179 reads)
T G G G G C C C A G A C T G A G C A C G C T T T G A T G G C A G A G G A A A G G
0.21% (120 reads)
T G G G G C C C A G A C T G A G C A C G C T T T T G A T G G C A G A G G A A A G
0.21% (116 reads)
K562 HEK3 Cas9-initiated HDR +1 CTT insertion
T G G G G C C C A G A C T G A G C A C - T G A T G G C A G A G G A A A G G A A G
17.15% (7256 reads)
T G G G G C C C A G A C T G A G C A C G T G A T G G C A G A G G A A A G G A A G
13.86% (5864 reads)
T G G G G C C C A G A C T G A G C A C G C T T T G A T G G C A G A G G A A A G G
10.55% (4461 reads)
T G G G G C C C A G A C T G A G C A - - T G A T G G C A G A G G A A A G G A A G
9.72% (4111 reads)
T G G G G C C C A G A C T G A G C A C G - - A T G G C A G A G G A A A G G A A G
5.14% (2174 reads)
T G G G G C C C A G A C T G A G C A C G G T G A T G G C A G A G G A A A G G A A
4.77% (2016 reads)
T G G G G C C C A G A C T G A G C A C G - G A T G G C A G A G G A A A G G A A G
3.82% (1616 reads)
T G G G G C C C A G A C T G A G C A C G T T G A T G G C A G A G G A A A G G A A
3.17% (1342 reads)
T G G G G C C C A G A C T G A - - - - - - - - T G G C A G A G G A A A G G A A G
1.92% (814 reads)
T G G G G C C C A G A C T G A G C A C G A T G A T G G C A G A G G A A A G G A A
1.63% (689 reads)
T G G G G C C C A G A C T G A G C A C G - - - - - - C A G A G G A A A G G A A G
1.26% (535 reads)
T G G G G C - - - - - - - - - - - - - - T G A T G G C A G A G G A A A G G A A G
0.78% (329 reads)
T G G G G C C C A G A C T G A G C A - - - - - - - - - - G A G G A A A G G A A G
0.61% (257 reads)
T G G G G C C C A G A C T G A G C A C G C T G A T G G C A G A G G A A A G G A A
0.59% (250 reads)
T G G G G C C - - - - - - - - - - - - - T G A T G G C A G A G G A A A G G A A G
0.56% (237 reads)
T G G G G - - - - - - - - - - - - - - - T G A T G G C A G A G G A A A G G A A G
0.52% (219 reads)
T G G G G C C C A G A C T G A G C A C G - - - - - - - - - A G G A A A G G A A G
0.47% (197 reads)
T G G G G C C C A G A C T G A G C A C G - - - - - - - A G A G G A A A G G A A G
0.43% (181 reads)
T G G G G C C C A G A C T G A G C A C G - - - - G G C A G A G G A A A G G A A G
0.41% (173 reads)
T G G G G C C C A G A C T G A G - - - - - - - - - - - - - - - G A A A G G A A G
0.40% (169 reads)
T G G G G C - - - - - - - - - - - - - - - - - - - - - A G A G G A A A G G A A G
0.38% (159 reads)
T G G G G C C C A G A C T G A G C A C G - - - - - - - - - - - G A A A G G A A G
0.37% (156 reads)
T G G G G C C C A G A - - - - - - - - - - - - T G G C A G A G G A A A G G A A G
0.35% (148 reads)
T G G G G C C C A G A C T G A G C A C G - - - - - - - - - - - - A A A G G A A G
0.35% (146 reads)
T G - - - - - - - - - - - - - - - - - - - - A T G G C A G A G G A A A G G A A G
0.31% (132 reads)
T G G G G C C C A G A C T G A G C T T - T G A T G G C A G A G G A A A G G A A G
0.30% (125 reads)
T G G G G C C C A G A C T G A G C A C G - - - - - - - - - - - - - - - - - A A G
0.30% (125 reads)
T G G G G C C C A G A C T G A G C A C G - - - - - - - - G A G G A A A G G A A G
0.29% (122 reads)
T G G G G C C C A G A C T G A G C A C G - - - - - - - - - - - - - - - - G A A G
0.29% (121 reads)
T G G G G C C C A G A - - - - - - - - - - - - - - - - - - - G G A A A G G A A G
0.27% (114 reads)
T G G G G C C C A G - - - - - - - - - - T G A T G G C A G A G G A A A G G A A G
0.26% (112 reads)
T G G - - - - - - - - - - - - - - - - - T G A T G G C A G A G G A A A G G A A G
0.26% (112 reads)
T G G G G C C C A G A C T G A G C A C G T G T G A T G G C A G A G G A A A G G A
0.26% (110 reads)
- - - - - - - - - - - - - - - - - - - - - - A T G G C A G A G G A A A G G A A G
0.24% (100 reads)
T G G G G C C C A G A C T G - - - - - - T G A T G G C A G A G G A A A G G A A G
0.23% (96 reads)
T G G G G C C C A G A C T G A G - - - - T G A T G G C A G A G G A A A G G A A G 0.23% (96 reads)
T G G G G C C C - - - - - - - - - - - - T G A T G G C A G A G G A A A G G A A G 0.22% (93 reads)
T G G G G C C C A G A C T G A G C A C G - - - - - G C A G A G G A A A G G A A G 0.21% (87 reads)

Extended Data Fig. 10 | See next page for caption.

Article
Extended Data Fig. 10 | PE3 activity in human cell lines and comparison of
PE3 and Cas9-initiated HDR. a, Prime editing in K562 (leukaemic bone
marrow), U2OS (osteosarcoma), and HeLa (cervical cancer) cells.
b–e, Efficiency of generating the correct edit (without indels) and indel
frequency for PE3 and Cas9-initiated HDR in HEK293T cells (b), K562 cells
(c), U2OS cells (d), and HeLa cells (e). Each bracketed editing comparison
installs identical edits with PE3 and Cas9-initiated HDR. Non-targeting controls
are PE3 and a pegRNA that targets a non-target locus. (f) Control experiments
with non-targeting pegRNA + PE3, and with dCas9 + sgRNA, compared with
wild-type Cas9 HDR experiments confirming that ssDNA donor HDR template,
a common contaminant that artificially elevates apparent HDR efficiencies,
does not contribute to the HDR measurements in a–d. g, Example HEK3 site
allele tables from genomic DNA samples isolated from K562 cells after editing
with PE3 or with Cas9-initiated HDR. Alleles were sequenced on an Illumina
MiSeq and analysed using CRISPResso243. The reference HEK3 sequence from
this region is at the top. Allele tables are shown for a non-targeting pegRNA
negative control, a +1 CTT insertion at HEK3 using PE3, and a +1 CTT insertion at
HEK3 using Cas9-initiated HDR. Allele frequencies and corresponding Illumina
sequencing read counts are shown for each allele. All alleles observed with
frequency ≥0.20% are shown. Mean ± s.d. of n = 3 independent biological
replicates.
 a b
Insertions (1,844 total) Duplications (6,154 total)
100 100 100 100

% of pathogenic duplications
% of pathogenic insertions

Cumulative frequency (%)

Cumulative frequency (%)

80 80 80 80
in ClinVar

in ClinVar
60 60 60 60

40 40 40 40

20 20 20 20

0 0 0 0
0 5 10 15 20 25 0 5 10 15 20 25
Insertion length (bp) Duplication length (bp)

c d
Deletions (19,706 total) Indels (1,530 total)
100 100 100 100
% of pathogenic deletions

Cumulative frequency (%)

Cumulative frequency (%)

% of pathogenic indels
80 80 80 80
in ClinVar

in ClinVar
60 60 60 60

40 40 40 40

20 20 20 20

0 0 0 0
0 5 10 15 20 25 0 5 10 15 20 25
Deletion length (bp) Indel length (bp)
Extended Data Fig. 11 | Distribution by length of pathogenic insertions, reported indels (single variants that include both insertions and deletions
duplications, deletions, and indels in the ClinVar database. The ClinVar relative to the reference genome) were calculated by determining the number
variant summary was downloaded from NCBI on 15 July 2019. The lengths of of mismatches or gaps in the best pairwise alignment between the reference
reported insertions, deletions, and duplications were calculated using and alternate alleles. a, Length distribution of insertions. b, Length
reference and alternate alleles, variant start and stop positions, or appropriate distribution of duplications. c, Length distribution of deletions. d, Length
identifying information in the variant name. Variants that did not report any of distribution of indels.
the above information were excluded from the analysis. The lengths of
 nature research | reporting summary
Corresponding author(s): David R. Liu
Last updated by author(s): Oct 8, 2019

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Methodology
Sample preparation 2.5 x 104HEK293T cells grown in the absence of antibiotic were seeded on 48-well poly-D-lysine coated plates (Corning). 16-24 h
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