nature biotechnology

Article https://doi.org/10.1038/s41587-024-02171-w

Genome engineering with Cas9 and AAV

repair templates generates frequent
concatemeric insertions of viral vectors

Received: 14 September 2022
Accepted: 8 February 2024
Published online: xx xx xxxx
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Fabian P. Suchy 1,2,10 , Daiki Karigane 1,3,4,5,10, Yusuke Nakauchi 1,3,4,
Maimi Higuchi1,2, Jinyu Zhang1,2, Katja Pekrun 2,6, Ian Hsu 1,2,7,
Amy C. Fan 1,3,4,8, Toshinobu Nishimura 1,2, Carsten T. Charlesworth1,2,
Joydeep Bhadury 1,2, Toshiya Nishimura1,2, Adam C. Wilkinson 1,2,7,
Mark A. Kay 2,6, Ravindra Majeti 1,3,4,10 & Hiromitsu Nakauchi 1,2,9,10

CRISPR–Cas9 paired with adeno-associated virus serotype 6 (AAV6) is

among the most efficient tools for producing targeted gene knockins. Here,
we report that this system can lead to frequent concatemeric insertions of
the viral vector genome at the target site that are difficult to detect. Such
errors can cause adverse and unreliable phenotypes that are antithetical
to the goal of precision genome engineering. The concatemeric knockins
occurred regardless of locus, vector concentration, cell line or cell type,
including human pluripotent and hematopoietic stem cells. Although these
highly abundant errors were found in more than half of the edited cells,
they could not be readily detected by common analytical methods. We
describe strategies to detect and thoroughly characterize the concatemeric
viral vector insertions, and we highlight analytical pitfalls that mask their
prevalence. We then describe strategies to prevent the concatemeric
inserts by cutting the vector genome after transduction. This approach is
compatible with established gene editing pipelines, enabling robust genetic
knockins that are safer, more reliable and more reproducible.

Genome engineering promises curative therapies for a multitude
of genetic conditions, such as cystic fibrosis, epidermolysis bul-
losa and most blood disorders, including sickle cell disease1–3. Still,
the tools available to genome engineers are imperfect; there is
room to increase editing efficiency, decrease off-target effects and
improve on-target fidelity4. The combination of CRISPR–Cas9 and
adeno-associated virus serotype 6 (AAV6) has proven to be highly
efficient for producing site-specific gene modifications5–8. As a virus,
AAV has evolved to deliver DNA in a less toxic and more efficient
manner than most other transfection protocols9. Typically, Cas9
ribonucleoprotein (RNP) is electroporated into cells and induces
a double-stranded break at the genomic target site, whereas AAV6
delivers single-stranded DNA repair templates into the nucleus.
The cell’s homology-directed repair machinery then repairs the

Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA. 2Department of Genetics,
1

Stanford University School of Medicine, Stanford, CA, USA. 3Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA. 4Department
of Hematology, Stanford University School of Medicine, Stanford, CA, USA. 5Japan Society for the Promotion of Science, Tokyo, Japan. 6Department
of Pediatrics, Stanford University, Stanford, CA, USA. 7MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK. 8Immunology
Graduate Program, Stanford University School of Medicine, Stanford, CA, USA. 9Distinguished Professor Unit, Division of Stem Cell Therapy, Institute
of Medical Science, University of Tokyo, Tokyo, Japan. 10These authors contributed equally: Fabian P. Suchy and Daiki Karigane; Ravindra Majeti and
Hiromitsu Nakauchi. e-mail: fsuchy@stanford.edu; rmajeti@stanford.edu; nakauchi@stanford.edu

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Article
Cas9-induced break using the AAV6-delivered DNA as a template.
This approach has been used to make both small changes and large
insertions in the genome of cells in vitro and in vivo10. The safety
of AAV is well studied as it has been used in over 100 clinical trials
worldwide11 for multiple purposes, including gene correction and
enzyme replacement therapy.
The use of AAV6 to deliver a repair template has boosted gene
knockin rates approximately 100-fold compared to plasmid transfec-
tion in various cell types. However, we found inconsistent results when
comparing PCR-based genotyping methods, which raised questions
about the fidelity of our targeted gene modifications. After developing
new strategies to detect more complex and unexpected genotypes, our
preliminary data suggested that half of the gene-edited cells contained
target-site concatemeric knockins of the AAV genome. Although other
groups reported anomalous insertions of AAV into Cas9 cut sites, those
analyses used PCR preamplification12,13. In contrast, the highly abun-
dant concatemeric knockins that we describe here have a distinct struc-
ture from previously reported anomalies, cannot be readily detected
by common PCR genotyping strategies and are more abundant than
other precision gene editing errors. In preclinical studies, concatemeric
knockins could obscure conclusions and hinder reproducibility. In
clinical studies, they could produce genetic knockouts or other unex-
pected phenotypes, yielding unintended and potentially dangerous
outcomes. A comprehensive analysis of the frequency and potential
consequences of concatemeric knockins has never been performed
because these anomalies and their abundance are not readily quanti-
fied by conventional methods.
In the present study, to improve Cas9/AAV6-mediated gene edit-
ing, we (1) developed and validated detection strategies to quantify
complex genotypes while showing why AAV-mediated concatemeric
insertions are not detected by common analytical methods; (2) report
here the frequency of concatemeric insertions at multiple loci of human
pluripotent stem cells (PSCs) and hematopoietic stem and progeni-
tor cells (HSPCs); and (3) developed an approach to greatly decrease
the prevalence of concatemeric knockins, with minimal change to
established preclinical and clinical gene editing pipelines. Using our
optimized method, we decreased the prevalence of unwanted con-
catemeric knockins by approximately 90%, with a minimal change in
knockin rate.
Results
Additional insertions detected by digital PCR
Because Cas9 RNP electroporation followed by AAV6 transduction
enables high-efficiency editing, knockins can be created without
selection, and purified populations can be isolated by subcloning
and genotyping (Fig. 1a). Employing this method, CRE was knocked
in to human induced PSCs (iPSCs) at the 3′ end of the CD14 locus, and
36 subclones were expanded. DNA was extracted from the subclones
and analyzed by three common PCR methods (Fig. 1b,c and Extended
Data Fig. 1a–d) to determine if the knockins were biallelic, monoallelic
or wild-type. Three-primer in–out PCR consistently reported the same
genotype when analyzing either the left (PCR-L) or the right (PCR-R)
side of the editing site. However, two-primer PCR, which is designed
to amplify the full region on both sides of the editing site (PCR-F),
was not concordant with PCR-L and PCR-R. In eight of 36 samples,
PCR-F was missing the upper (KI: knockin) band shown in PCR-L and
PCR-R (Fig. 1d and Extended Data Fig. 1e). To further elucidate the
genotype, an alternate method was developed, which uses droplet
digital PCR (ddPCR) to count wild-type alleles (no KI) and CRE (KI)
alleles (Extended Data Fig. 1f,g). Because each cell contains two cop-
ies of the CD14 locus, the sum of no KI and KI alleles should equal two
per cell. This strategy found that more than half of the edited cells
contained additional copies of CRE at integer intervals (Fig. 1e and
Extended Data Fig. 1h,i). Additional copies were detected in all samples
with discordant PCR analyses.
Nature Biotechnology
https://doi.org/10.1038/s41587-024-02171-w
Additional insertions are concatemers
As is commonplace, the ddPCR analysis in Fig. 1e was performed with
restriction enzymes to fragment the genomic DNA in a controlled man-
ner. When rerunning the reaction without fragmentation, the number
of detected additional insertions was greatly diminished in most of the
subclones (Extended Data Fig. 1i). Because ddPCR is similar to limiting
dilution assays, linked regions of the genome partition together in the
same droplet when the DNA is not fragmented. Therefore, concate-
meric knockins would be counted only once in non-fragmented DNA
(Extended Data Fig. 1j,k). This suggests that the additional insertions are
concatemeric in all subclones except number 12 (Extended Data Fig. 1i).
Southern blots can measure the size of large regions of DNA with-
out PCR amplification. Because the concatemers were not readily
detected by PCR, 11 subclones with various ddPCR genotypes were
further expanded and analyzed by Southern blot (Fig. 1f and Extended
Data Fig. 1l,m). The Southern blot revealed that many samples had
larger knockin bands at regular intervals. As expected, most sam-
ples had only one or two bands; however, sample 12 displayed a third
band of irregular size, which corresponds to the off-target insertion
detected by ddPCR. The Southern blot analysis was 100% concordant
with the ddPCR allele counting strategy. Collectively, these data sug-
gest that concatemeric knockins frequently occur when using Cas9/
AAV-mediated genome editing. These concatemers are difficult to
detect by classic PCR genotyping strategies, but they are revealed by
Southern blots and ddPCR after controlled genomic fragmentation
(Fig. 1g). Because Southern blots generally require multiple days of
preparation and consume more than 1,000-fold more starting mate-
rial than ddPCR, ddPCR analyses were used for subsequent genotyp-
ing. Repeat experiments were performed at the AAVS1 locus and the
genotype analyzed by PCR and ddPCR. Results similar to those at the
CD14 locus were seen at the AAVS1 locus, with concatemeric insertions
occurring in 39% of CD14 subclones and 36% of AAVS1 subclones (Fig. 1h
and Extended Data Fig. 2).
Concatemers contain ordered repeats of full vector genome
The band sizes on the CD14 Southern blot were measured to deter-
mine the length of the concatemers (Fig. 2a). Bands were spaced at
concise intervals, with an average distance of 2.6 kb between groups
of concatemeric bands. Because this is the same size as the full AAV6
vector genome used to deliver the knockin template, multiple inser-
tions could result from linked vector genomes as shown in Fig. 2b.
PCR was attempted across the potential head-to-tail concatemeric
junction (Extended Data Fig. 3a–c). Indeed, bands appeared only in
samples that contained concatemeric knockins. However, the PCR was
inefficient and resulted in two weak bands (Fig. 2c). Sanger sequenc-
ing revealed that the bottom band was missing the inverted terminal
repeats (ITRs) entirely, containing a perfect linkage of opposing left
and right homology arms. Sanger sequencing failed on the upper band,
so it was sequenced with nanopore technology instead. This revealed
a unique chimeric ITR composed of two back-to-back ITRs missing
their distal ends (Extended Data Fig. 3d and Supplementary File 1).
Reamplification of gel-extracted upper and lower bands resulted in
only the lower bands, suggesting that the lower band is an artifact of
PCR that occurs with an increasing number of PCR cycles. No bands
were seen when using primer combinations to detect head-to-head or
tail-to-tail concatemers (Extended Data Fig. 3b,c). Collectively, these
data suggest that (1) concatemeric knockins are joined by the viral
vector ITRs, which creates a distinct chimeric ITR; (2) PCR amplifica-
tion and subsequent sequencing across the chimeric ITR is difficult;
and (3) the concatemers exist primarily in a head-to-tail orientation.
We next sought to test the regularity of the concatemeric knock-
ins. Because the full-length concatemeric KI cannot be readily PCR
amplified and directly sequenced, ddPCR was used to detect the stoi-
chiometry of DNA segments corresponding to regions of the vector
genome. For each additional CRE, there was also an additional left
Article https://doi.org/10.1038/s41587-024-02171-w

a
Knockin Subclone Genotype

Cas9 WT Mono Bi

Locus: LHA RHA

No No No
KI KI KI KI KI KI

AAV6: LHA Insert RHA

b PCR-L (left) PCR-R (right) PCR-F (full) c

o
F R2 F2 R F R

on
T
W

Bi
M
KI:
KI
F R1 F1 R F R
No No KI
KI:

d S9 S11 S26 S10 S8 S13 S17 g

R
R
L

PC
R-
R-

R-
PC
PC

PC

dd

SB
PCR-L KI
9
No KI

Individual subclones
1
2
4
6
PCR-R KI 7
15
No KI 18
19
20
WT Mono Bi Mono Bi Mono Bi 22
23
25
29
KI 32
PCR-F 35
10
No KI 13
21
WT Mono Bi WT Bi WT ? 30
31
11
5
e 34
12
26
5 3
Extra copies/cell

33
KI (CRE) 8
27
Total copies/cell

4 2 36
3
No KI 28
17
3 1 24
16
14
2 0
WT Mono Bi
1
Irregularity
0
WT Mono Bi Mono Bi Mono Bi
+1 +1 +2 +3
h CD14CRE AAVS1Ubc-CRE
100%
f
KI + 2 80%
KI + 1 60%

KI 40%
39% 36%
20%
No KI
0%
WT Mono Bi Mono Bi Mono Bi
L

L
R

R
R

R
F

F
R-

R-
PC

PC
R-

R-
R-

R-

+1 +1 +2 +3
PC

PC
PC

PC
PC

PC
dd

dd

Fig. 1 | Concatemeric knockins detected by digital PCR. a, Pipeline schematic
for Cas9/AAV-mediated gene editing in PSCs. AAV6 vector ITRs are indicated
as hairpins in orange. Bi, biallelic knockin; Mono, monoallelic knockin; WT,
wild-type (no knockins). b, Schematic showing three PCR genotyping strategies.
PCR-L = three-primer in–out PCR performed spanning the left homology arm.
PCR-R = three-primer in–out PCR performed spanning the right homology
arm. PCR-F = two-primer PCR with primers located outside of both homology
arms. c, Schematic showing expected gel electrophoresis banding patterns and
corresponding genotypes from PCR reactions in b. KI, band corresponding to
knockin allele. No KI, band corresponding to allele without a knockin. d, PCR
genotype of select subclones after knocking in CRE in the CD14 locus in human
PSCs. Sample number is indicated on the top row, interpreted genotype on the
bottom row. Red indicates samples with discordance between PCR-L/PCR-R and
PCR-F. e, Digital PCR genotype of select samples indicated in d. Two copies per
cell are expected (left axis). Extra copies are shown on the right axis, interpreted
genotype at bottom. + indicates additional insertions. f, Southern blot of select
samples indicated in d. g, Comparison of PCR-L, PCR-R, PCR-F, ddPCR and
Southern blot genotype after knocking in CRE into the CD14 locus in human PSCs.
Each row indicates an individual sample; sample number is indicated on the left.
Red indicates unexpected result due to incorrect band size, missing information
(for example, no band) or additional insertions. SB, Southern blot; WT, wild-
type. h, Summary of genotype frequencies detected by different methods after
knocking in CRE into the CD14 locus (left) and Ubc-CRE into the AAVS1 locus
(right). Bar colors are the same as g.

homology arm and right homology arm detected at a 1:1 ratio (Fig. 2d and sample 14, which had an end-joining mediated on-target knockin
and Extended Data Fig. 3e,f). This was true for all samples except sample verified by PCR and sequencing. ddPCR failed to detect the ITR in all
12, which had a partial off-target insertion verified by Southern blot, samples except sample 14 (Extended Data Fig. 3i; ITR −RE), suggesting

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Article https://doi.org/10.1038/s41587-024-02171-w

a b Linked viral genomes d 10

16 1.46 kb 2.55 kb LHA

Extra LHA, RHA, ITR KI (copies/cell)

14 LHA CRE RHA LHA CRE RHA RHA
KI+4 8
+3.2 kb ITR
12
Genomic locus
SB band size (kb)

KI+3
10 ~0.8 kb 6
+2.6 kb Concatemeric knockin
8 KI+2 CRE RHA LHA CRE
+2.6 kb 4
6
KI+1
c WT M M + Off M + 1 M + 2 Bi Bi + 1 Bi + 2 Bi + 3 Bi + 4 Bi + 5
4 +2.4 kb
0.85 kb
KI 0.65 2
2 +1.4 kb 0.50
No KI 0.40
0 S9 S11 S12 S10 S13 S26 S8 S36 S17 S27 S24 L
1 2 3 4 5
Extra CRE KI (copies/cell)
e
Dual knockin Sort & subclone Analyze
Cas9 Bi Mono+ h
mCherry

GFP = 1 GFP = 4 GFP = 1 GFP = 1

mCh = 5 mCh = 2 mCh = 8 mCh = 3
LHA Ubc-GFP RHA KI KI KI + 1 No KI

High gate
LHA Ubc-mCh RHA
GFP
GFP = 2 GFP = 4 GFP = 3 GFP = 2
f 5 g mCh = 3 mCh = 4 mCh = 1 mCh = 5
10
n = 48
Colonies w/ concatemers

100%

4
80%
10
GFP = 1 GFP = 1 GFP = 1 GFP = 1
60%
mCh = 1 mCh = 1 mCh = 1 mCh = 1
High gate
40% Low gate
10
3
(13.3%)
n = 24
Low gate 20%
(7.7%) GFP = 1 GFP = 1 GFP = 1 GFP = 1
0% mCh = 1 mCh = 1 mCh = 1 mCh = 1
mCherry

0 High
Highgate
gate Low
Lowgate
gate
GFP GFP or mCh mCh

0
3
10 10
4
10
5
GFP mCh
GFP

Fig. 2 | Characterization of concatemeric knockins. a, Consolidated plot of all additional insertions of CRE per cell. The y axis indicates ITRs per cell (yellow),
Southern blot (SB) band sizes. Samples are 11 selected subclones from CRE knockin additional left homology arms per cell (LHA, purple) and additional right
to the CD14 locus in human PSCs. Average size of each group is indicated by dashed homology arms per cell (RHA, green). e, Schematic showing dual knockin
lines. Red band indicates off-target insertion in subclone 12. b, Schematic showing and double selection. f, Flow cytometry plot of dual knockin as indicated in e.
viral vector genome size and theoretical arrangement of concatemeric knockin. Performed on human PSCs at the TET2 locus, 5 d after editing. g, Graph indicating
Red arrows indicate primers used for junction PCR in c. c, Junction PCR of 11 subclones with at least one concatemeric knockin. Subclones were sorted and
selected samples (same as a). Primers span concatemer linkage site. Sample ID is expanded from high gate and low gate indicated in f (n = 48 high gate; n = 24 low
shown on bottom. Red indicates that sample had off-target insertion as indicated gate). Blue indicates that the subclone had at least one concatemeric knockin with
by ddPCR. Genotype (ddPCR) is listed on top. B, biallelic knockin; M, monoallelic GFP and mCherry inserted in the same allele (GFP and mCherry linkage). Gray
knockin; WT, wild-type (no knockins). + indicates the number of additional indicates all other concatemeric knockins. h, Representative fluorescent images
concatemeric insertions. +Off indicates off-target insertion(s). d, Plot comparing of GFP and mCherry in subclones from f. All 16 images were processed identically.
the copy number of concatemer segments measured by ddPCR. Eleven subclones GFP and mCherry copies per cell indicated on image (detected by ddPCR).
were analyzed (same as c) after restriction enzyme digestions. The x axis indicates Gray scale bar (lower right) indicates 200 µm.

that the chimeric ITR formed in concatemeric knockins is more PCR Concatemers affect gene expression
resistant than the ITR found in end-joining mediated knockins that If ITRs are the drivers of concatemerization, then concatemeric knock-
were previously reported12–14. ins should occur between two viral vector particles carrying different
Because PCR inefficiencies can be due to nearby DNA secondary DNA sequences. To test this, a dual knockin of mCherry and GFP was
structures, ITR quantification was repeated with restriction enzymes attempted in iPSCs at the TET2 locus (Fig. 2e). Five days after editing,
that cut within the chimeric ITR, adjacent to the amplicon (Extended the cells were analyzed and sorted by flow cytometry for subcloning
Data Fig. 2d). This strategy led to more efficient qPCR and ddPCR detec- (Fig. 2f). The single-positive quadrants (that is, mCherry only and GFP
tion of the ITR (Extended Data Fig. 3g–i), revealing that two ITR regions only) appeared bimodal, which likely corresponds to a monoallelic or
exist for each additional insertion of CRE (Fig. 2d), which agrees with biallelic knockin of a single color. The double-positive quadrant had a
the schematic shown in Fig. 2b. To ensure that the detected ITRs were large, polydisperse region. This heterogenous gene expression could
not episomal, a ddPCR linkage analysis was performed to determine be due to concatemeric knockins. Twenty-four colonies were sorted
whether the genomic locus was linked to the ITRs (Extended Data and expanded from the dimmer, more homogeneous cluster (Fig. 2f,
Fig. 3j). All loci with concatemers had ITRs linked to one or both CD14 low gate), and 48 colonies were sorted and expanded from the brighter,
alleles (Extended Data Fig. 3k). These data prove that the concatemeric polydisperse cluster (Fig. 2f, high gate). ddPCR linkage analysis was
knockins have ITR sequences that are integrated into the genomic used to analyze the genotype (Extended Data Fig. 4a,b), revealing
locus, and these integrated chimeric ITRs are hidden without careful that 46 of 48 high gate subclones contained concatemers (Fig. 2g). In
restriction enzyme digestion. contrast, only four of 24 low gate subclones contained concatemers.

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Article https://doi.org/10.1038/s41587-024-02171-w

a d NS NS e
Concatemer Locus Cell line n
120% ** *
LHA Insert RHA LHA Insert RHA TET2 PT-iPSC 64

Relative KI efficiency
100%
AAVS1 PT-iPSC 79
Genomic locus
80%
AAVS1 H9 57
60%
LHA Insert RHA LHA Insert RHA
AAVS1 WT-iPSC 58
40%
Delinked monomers
20% HBB PT-iPSC 65

b Internal ITR cut (IC) Distal cut (DC) 0% HBB H9 67

NC IC DC NC IC DC
Virus: LHA Insert RHA LHA Insert RHA Positive Double-pos. RUNX1 H9 68
cells cells
Cas9
Cas9
Cas9
f g
Locus: 100% **** 60%
*** Positive cells
50% KI alleles w/
59%
c

Colonies w/ concatemers
80% concatemer
5
No cut (NC) ITR cut (IC) Distal cut (DC)
10 40%
15.7% 25.5% 16.2% 17.3% 19.9% 16.1%
60%

10
4 30%

40%
13% 20%
3
10
4.8%
20%
mCherry

10%

0 31.8% 27.0% 35.2% 31.3% 38.4% 25.5% 0% 0%

3 4 5 3 4 5 3 4 5 NC IC DC 3,162 1,000 316 100 32 10
0 10 10 10 0 10 10 10 0 10 10 10
GFP MOI

Fig. 3 | Frequency and prevention of concatemers in human PSCs.
a, Schematic showing delinking of AAV vector genomes. b, Schematic showing two
methods to remove vector ITRs after transduction using Cas9. c, Flow cytometry
plot 5 d after double knockin of Ubc-GFP and Ubc-mCherry into TET2 locus in
PT-iPSCs. d, Change in knockin efficiency as determined by flow cytometry. IC and
DC are shown relative to NC. n = 7 (seven test groups shown in e). ** and * indicate
P = 0.0008 and P = 0.01, respectively. NS, no significance (P > 0.05). P values
are from a two-sided paired t-test. e, Table of different loci and cell lines tested;
legend for f. n indicates the number of subclones analyzed in each group. f, Clonal
analysis of concatemer frequency after double knockin (determined by ddPCR).
Comparison of NC, IC and DC methods. Loci and cell lines shown in e. 16–28
colonies were analyzed per datapoint; n = 458 total. Mean is indicated above the
bar in bold. *** and **** indicate P = 2 × 10−6 and P = 2 × 10−7, respectively. P values are
from a two-sided t-test. g, Bulk analysis of Ubc-GFP knocked in to HBB locus in H9
embryonic stem cells (ESCs) at various vector dilutions. GFP+ cells (determined by
flow cytometry) are indicated by the gray line. The percent of KI alleles that contain
a concatemeric insertion (determined by ddPCR) is indicated by the red line. Error
bars indicate 1 s.d.; center indicates the mean.

Fluorescence imaging revealed that high gate subclones were brighter the same time as the RNP that cuts the target gene. For the second
and had more well-to-well fluorescence variability than the low gate strategy, the 23-bp target sequence recognized by the RNP that cuts
subclones, which was made more apparent with uniform image process- the genomic locus was inserted into the vector genome on both ends
ing of the entire set of subclones (Fig. 2h and Extended Data Fig. 4c). flanking the homology arms, such that the gene-targeting RNP would
The mean fluorescence intensities (MFIs) correlated strongly with the also make two distal cuts (DCs) in the vector genome and remove the
total number of insertions (Extended Data Fig. 4d,e). Linkage analysis ITRs. IC and DC methods were compared to the original method (no
revealed that most of the low gate subclones with concatemers had a cut (NC)) in three PSC lines and four loci, including the clinically rel-
monoallelic knockin genotype, such that both mCherry and GFP were evant HBB locus. For all conditions, double-fluorescent knockins were
inserted into a single allele, whereas the other allele did not contain a selected as previously shown in Fig. 2e. Flow cytometry revealed a
knockin. This is particularly problematic for gene editing strategies clear decrease in polydispersity when using IC or DC methodology,
that employ dual knockins to achieve a selectable biallelic genotype particularly in the double-positive quadrant (Fig. 3c and Extended
because some cells will still contain an unedited allele. Overall, 50 of Data Fig. 5a–h). The overall knockin rate (that is, percent of fluorescent
72 subclones had concatemeric knockins, of which 21 of 50 had at least cells) for NC ranged from 27% to 79%, which is similar to previously
one mCherry and one GFP linked in the same allele. These data show published values5. IC and DC treatments resulted in minor decreases
that concatemeric knockins can also occur between vector particles in average overall editing efficiencies by 19% and 9%, respectively;
carrying different DNA. Notably, concatemeric knockins may greatly however, the reduction in the double-positive cells was more pro-
change the level of gene expression. nounced (42% and 23% decreases, respectively; Fig. 3d and Extended
Data Fig. 5i–k). From each condition (Fig. 3e), 16–28 subclones (458
ITR removal prevents concatemers in PSCs total) were sorted and expanded from the double-positive population.
If the ITRs are driving the concatemeric knockins, then removal of ITRs ddPCR genotyping revealed that an average of 59% of the subclones
should result in more monomeric knockins (Fig. 3a). Two strategies in the NC group had concatemeric knockins (Fig. 3f). This decreased
were designed that use Cas9 RNPs to remove the viral vector ITRs after to 13.2% and 4.8% for the IC and DC groups, respectively. These data
transduction with minimal modification to gene editing pipelines. For show that concatemeric knockins occur at high frequency in human
the first strategy, a single guide RNA (sgRNA) was designed to make PSCs, regardless of the cell line or locus. Notably, post-transduction
an internal cut (IC) within the ITR at an endogenous PAM site (Fig. 3b). removal of the viral vector ITRs significantly reduces the frequency
This sgRNA is complexed with Cas9 and electroporated into cells at of concatemeric knockins.

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Article
Rate of concatemers unaffected by viral vector concentration
It is known that the multiplicity of AAV infection (MOI) will affect
knockin rate8, but how MOI affects the rate of concatemeric knockins
is unknown. For this analysis, Ubc-GFP was knocked in to the HBB locus
at various MOI dilutions ranging from approximately 3,000 to 10. In
this case, MOI refers to the number of vector genomes (determined by
ddPCR titer) added per cell. To avoid the high sampling error that can
be prevalent in small-population-size clonal analyses, a ddPCR linkage
analysis strategy was developed to detect concatemeric alleles in a
bulk, mixed population (Extended Data Fig. 6a,b).
As expected, flow cytometry revealed that the percent of posi-
tive cells decreased from 56% to 2% as MOI was reduced (Fig. 3g
and Extended Data Fig. 6c). The positive cells were then sorted and
expanded in each MOI group without subcloning. Bulk allele analy-
sis showed that approximately 35% of the knockin alleles were con-
catemeric at all MOIs (Fig. 3g and Extended Data Fig. 6d–h). This was
repeated in male PSCs at the IL2RG locus, located on the X chromosome,
which yielded similar results (Extended Data Fig. 6i–m). In both cases,
the concatemeric insertions contained an average of 3–4 repeats at all
MOIs. Collectively, these data suggest that the concatemeric knockin
rate is constant in human PSCs, regardless of locus, cell line, MOI or
number of genomic target sites. If an allele has a knockin, there is a 35%
chance that it is concatemeric. Thus, the theoretical frequency of at
least one concatemeric knockin occurring in a biallelically edited PSC
is 58%. This matches our empirical result from Fig. 3f.
ITR removal does not increase non-homologous end-joining
knockin
ITR removal changes the structure of the vector genome, potentially
altering its dynamics regarding the frequency of non-homologous end
joining (NHEJ)-mediated insertions. This could cause imprecise knock-
ins. To analyze this, in–out PCR was used to check for NHEJ-mediated
knockins at the TET2 locus in iPSC subclones with seemingly correct
ddPCR genotypes (that is, biallelic knockins without concatemers). All
samples except for one subclone from the IC group had normal band
sizes (Extended Data Fig. 7a–d). This indicates that NHEJ-mediated
knockin events are uncommon in all groups, and subcloning is not
an efficient method to measure their frequencies. As an alternative,
flow cytometry was used to measure NHEJ-mediated insertions by
repeating HBB and IL2RG knockin experiments while intentionally
mismatching the Cas9 RNP genomic target sites and vector homol-
ogy arms. When making a cut at the HBB locus, a vector that carried
Ubc-GFP with homology arms targeting the IL2RG locus was added. The
reciprocal experiment was also performed (Extended Data Fig. 7e,f).
When cutting the HBB locus, insertions occurred in 0.53% and 0.50%
of the cells in the NC and IC groups, respectively. When cutting the
IL2RG locus, insertions occurred in 0.15% and 0.08% of the cells in the
NC and IC groups, respectively. These data suggest that ITR removal
does not increase the frequency of unwanted NHEJ-mediated knockins.
Concatemers also occur in HSPCs
CD34+ blood cells contain both hematopoietic stem and progenitor
cells. Because the stem cell population (HSCs) is difficult to enrich from
HSPCs, HSPCs are the targets of multiple gene therapies15. To deter-
mine if concatemeric knockins occur in HSPCs, Cas9/AAV6-mediated
double-knockin experiments were repeated in human cord blood
(CB)-derived CD34+ HSPCs. Because clonal HSPC expansion is ineffi-
cient and the DC method appeared more effective than the IC method,
only NC and DC groups were compared. When performing knockins
at the TET2 and HBB loci, results were similar to those of PSCs, with an
average of 58% and 7.0% of the subclones having concatemeric knockins
in the NC and DC groups, respectively. The experiment was repeated
with male CB at the IL2RG locus (X chromosome), resulting in 38% and
1.4% of colonies containing concatemeric knockins in the NC and DC
groups, respectively (Fig. 4a–d and Extended Data Fig. 8a–l).
Nature Biotechnology
https://doi.org/10.1038/s41587-024-02171-w
Only a subset of the CD34+ HSPCs can enable long-term recon-
stitution of the blood after bone marrow transplantation. Therefore,
it is necessary to determine whether concatemeric insertions occur
in the functional stem cells or just the progenitor cells. To test this,
Ubc-GFP was knocked in to human CD34+ cells at the HBB locus, and
GFP+ cells were transplanted into immunodeficient mice a few days
later (Fig. 4a). After 4 months, bone marrow was collected from the
mice, and the human cells were isolated by flow cytometry. Because
transplanted blood progenitor cells generally exhaust after 12 weeks
in mice, the remaining human cells were likely derived from engrafted
human HSCs16. Bulk ddPCR analysis revealed that an average of 32% of
the edited human alleles had concatemeric knockins in the four mice
from the NC group. This dropped to below 5% for the DC group (Fig. 4e).
Notably, the average engraftment rate of edited cells was 0.22% and
2.5% for the NC and DC groups, respectively, suggesting that the DC
approach did not impair HSC engraftment (Extended Data Fig. 8m).
Collectively, these data demonstrate that Cas9/AAV6-mediated con-
catemeric knockins also occur at high frequency in human HSCs. Like
in iPSCs, this can be minimized by post-transduction ITR removal.
Discussion
Here we report an analytical blind spot in the detection of repeated
genetic sequences, particularly when linked by a region containing com-
plex secondary structures. Our ddPCR assays found that approximately
35% of Cas9/AAV6-mediated KI alleles contain at least one hidden
concatemeric insertion, regardless of loci or cell type. We developed
drop-in-ready methods that decrease the number of these abnormal
gene modifications by approximately 10-fold.
All data were generated using human HSCs14 and human PSCs17
because of their clinical relevance for cell and gene therapy. HSCs give
rise to all lineages of blood cells and can be collected from a patient’s
blood or bone marrow. The HSCs can then be genetically modified and
transplanted back into the patient to cure conditions such as sickle
cell disease3. In contrast, iPSCs can be derived from many different tis-
sues, including skin or blood. iPSCs can be expanded indefinitely, ena-
bling numerous and extensive genetic modifications in vitro. Because
iPSCs resemble one of the earliest lineages of cells during embryonic
development, patient-derived iPSCs can be differentiated into other
cell types and transplanted back into the patient. This has been a pro-
posed treatment for cystic fibrosis and epidermolysis bullosa1,2. Cas9/
AAV6-mediated gene editing has been performed on both HSCs and
iPSCs with remarkably high efficiencies5,6,8, and it is used in other clini-
cally relevant cell types, including CAR-T cells18.
Many common gene editing goals are susceptible to inconsist-
ent gene modifications. When creating repairs or knockins in coding
regions, concatemeric insertions could result in frameshift-induced
knockouts (Fig. 5a). A similar point is true if exchanging or adding
exons, particularly at the 5′ end of the gene. In other cases, it is essential
to create biallelic, selectable knockouts to ensure 100% gene disrup-
tion. As previously published, this can be performed by knocking in two
different selectable markers into the target gene, one in each allele19.
However, with concatemeric insertions, both selectable cassettes
could be knocked in to a single allele, whereas the other remains intact
(Fig. 5b). Concatemeric insertions can also cause problems related to
inconsistent and high levels of gene expression. This could be critical
for transgenic safeguards, such as inducible caspase-9 (ref. 20), which
usually require addition of a small molecule to induce dimerization
and initiate apoptosis. However, at high concentrations, inducible
caspase-9 can spontaneously dimerize and uncontrollably kill cells21.
This is more likely to occur with concatemeric insertions because they
often induce higher expression (Fig. 5c). In other cases, researchers
simultaneously knock in two different inserts into separate target
sites19. However, concatemerization could cause knockins to occur at
the wrong locus (Fig. 5d), leading to numerous downstream problems.
A final concern pertains to the ITR itself, which has been shown to act as
Article https://doi.org/10.1038/s41587-024-02171-w

a
CD34+ Sort Colony expansion Concatemer
HSPCs analysis

mCherry
3 weeks 5
4
3

GFP 2
1
0

Knockin

4 months
NC: LHA GFP, mCh RHA

DC: LHA GFP, mCh RHA Transplantation Human blood cells

b * * c
120% Locus KI Type n

TET2 GFP, mCh colony 42

100%
Relative KI efficiency

TET2 GFP, mCh colony 31

80%
HBB GFP, mCh colony 33
60%
HBB GFP, mCh colony 33
40%
IL2RG GFP colony 46
20%
IL2RG GFP colony 38

0% IL2RG GFP colony 34

NC DC NC DC
Positive Double-pos. HBB GFP Bulk (transplant) 4 mice
Positive
cells Double
cells pos.
HBB GFP Bulk (transplant) 4 mice

d TET2 & HBB colonies IL2RG colonies e Transplanted cells

80% 50%
*** * ***
40%
KI alleles w/ concatemer
Colonies w/ concatemer

60%

30%

40%

20%

20%
10%

0% 0%
NC DC NC DC NC DC

Fig. 4 | Frequency and prevention of concatemers in human HSPCs.
a, Schematic showing strategy for analyzing Cas9/AAV-mediated concatemeric
knockins in hSPCs. b, Change in knockin efficiency as determined by flow
cytometry. DC is shown relative to NC. Test groups are shown in c. n = 9 for
positive cells; n = 4 for double positive. * indicates P = 0.02. P values are from a
two-sided paired t-test. c, Table of different loci and cell lines tested; legend for
d and e. n indicates the number of subclones or mice analyzed in each group.
d, Clonal analysis of concatemer frequency after double knockin (left) and single
knockin (right) as determined by ddPCR. Comparison of NC and DC methods.
Loci and cell lines are shown in c. 15–23 colonies were analyzed per datapoint;
n = 258 total. *** and * indicate P = 0.0002 and P = 0.03, respectively. P values are
from a two-sided t-test. e, Bulk ddPCR concatemer analysis of recovered human
cells 4 months after HSPC transplantation. Before transplantation, HSPCs had
Ubc-GFP knocked in to the HBB locus. *** indicates P = 0.0004. P values are from a
two-sided t-test. Error bars indicate 1 s.d.; center indicates the mean.

a transcriptional regulator22,23. Therefore, a concatemeric knockin may
affect the expression of nearby genes, resulting in wildly unpredictable
phenotypes (Fig. 5e).
As we have shown, concatemeric knockins are difficult to detect
by classic PCR (Fig. 1d–h). ddPCR has been previously used to genotype
cells by adapting in–out PCR to quantify on-target knockins24. However,
in–out PCR must span a homology arm, resulting in long amplicons
that can be difficult to multiplex and detect by ddPCR. Additionally,
in–out PCR is not designed to detect concatemeric knockins, so an
alternative method is needed. In the present study, we developed three
types of ddPCR assays that can be used to simultaneously genotype and
detect concatemeric knockins: (1) ‘allele counting’, (2) ‘linkage analy-
sis’ and (3) ‘nuclease-mediated loss of linkage’. Each assay is modular
and has strengths in different research settings. For example, allele
counting with fragmentation is best for subcloned cells with no epi-
somal vector DNA, particularly if researchers want to perform only a
single run for easy screening. However, in bulk populations or samples
that contain episomal vector DNA, allele counting cannot be used to

Nature Biotechnology
Article https://doi.org/10.1038/s41587-024-02171-w

a Gene correction concatemeric alleles are spread across a variety of sizes (for example,
Insert KI + 1, KI + 2, KI + 3, etc.), resulting in a higher proportion of wild-type
and monomeric KI alleles in bulk samples. Because subcloning can
Gene knockout be technically challenging, it is often avoided. Third, concatemeric
Insert ITR Insert alleles are bigger than monomeric KI alleles. This can lead to PCR bias
and allelic dropout26,27. Therefore, strategies that do not require PCR
b Selectable biallelic knockout
preamplification across full concatemers are needed, such as Southern
blot or ddPCR. However, obtaining enough DNA for Southern blot
Ubc-GFP
analysis can be challenging, particularly in cells that do not expand well,
Ubc-mCh
such as HSPCs. Fourth, even if using ddPCR methodology designed to
One allele intact count insertion numbers in subcloned cells, concatemeric knockins
will not be detected unless first separated by the addition of nucleases
Ubc-GFP ITR Ubc-mCh
(Extended Data Fig. 1j,k). The DNA must also be in proper form (for
example, double stranded and properly annealed) for the nucleases to
cut. Fifth, the ITR itself is considerably more difficult to detect by PCR
c Low gene expression in its linked, chimeric form (Extended Data Fig. 3g–i, sample 10). This
Ubc-iCasp9 is particularly misleading because we can easily amplify the ITR region
when tittering the vector in its single-stranded DNA form. Addition-
High gene expression
ally, the ITR can be readily detected when inserted into the genome
Ubc-iCasp9 ITR Ubc-iCasp9 ITR Ubc-iCasp9 ITR Ubc-iCasp9
in an NHEJ-mediated manner (Extended Data Fig. 3g–i, sample 14).
However, to detect the chimeric ITR, restriction enzymes must be
d Multi-site editing added to dismantle nearby secondary structures. Sixth, although
Insert 1 fluorescent concatemeric knockins can be detected by variable fluo-
Insert 2 rescence intensities, the log-scaled axis visually attenuates this affect
when analyzed by flow cytometry. The use of contour maps further
Insert into wrong site minimizes the polydispersity caused by concatemeric knockins. In
Insert 1 ITR Insert 2 ITR Insert 1 subclones, clear differences can be seen in the fluorescence intensities
Insert 2 of multiple samples only if they are imaged and processed identically
(Extended Data Fig. 4c). The difference is less noticeable if using com-
e Knockin
mon ‘auto-contrast’ options. For these reasons, the high frequency of
Cas9/AAV6-mediated concatemeric knockins has not been reported.
Insert
We considered multiple strategies to prevent concatemeric
Altered nearby gene expression
knockins, including decreasing the MOI and performing intracellular
Insert ITR Insert removal of ITRs. As expected, lowering the MOI greatly diminished
the number of KI cells and shifted most edited cells from biallelic to
monoallelic genotypes. Low MOIs had no effect on the rate of concate-
meric KIs. Mechanistically, this suggests that viral vector concatemeric
Fig. 5 | Abnormal phenotypes with concatemeric knockins. a, Concatemeric
linkages occur faster than genomic insertion. This could be explored
knockins often cause frameshift mutations (indicated by black stripes), resulting
in future experiments by altering the interval between vector trans-
in gene knockout. b, Use of two reporters to make selectable biallelic knockouts
fails if both reporters are inserted in one allele. c, Large concatemers inserted in a
duction and Cas9 transfection while measuring the concatemer rate
single site can increase gene expression to unwanted levels. d, Multi-site genome at various intervals.
editing can result in knockin of template in the wrong locus. e, Enhancer activity It is also possible that concatemers may form within the viral
of ITR can affect nearby gene expression. vector particles before transduction. However, all vector genomes
containing Ubc-GFP and Ubc-mCherry are more than 3,200 bp long.
A single concatemeric vector genome would have to be larger than
detect concatemeric knockins. In contrast, linkage analysis can detect 6,400 bp, which is difficult to package in AAV. Additionally, many
concatemeric knockins in subcloned and bulk populations, even if a concatemeric repeats are more than 3 units long and some more than
moderate amount of episomal vector DNA or off-target insertions are 10, suggesting that prepackaging is not the cause of concatemeric
present. However, shortly after transduction with AAV, the amount of knockins. Finally, experiments were performed using two viral vec-
episomal vector DNA is too high and may fall outside the limits of reli- tors from different preparations, in which approximately half of the
able ddPCR linkage quantitation. In this case, the nuclease-mediated concatemeric insertions contained DNA from both vectors knocked in
loss of linkage strategy can be employed to quantify knockins and to one allele (Fig. 2g). This suggests that linkage occurs after transduc-
concatemeric inserts (Extended Data Fig. 3m, bottom). Although not tion, and it is unbiased between vectors originating from the same or
extensively discussed here, this unique assay uses site-specific nucle- different preparations.
ases (for example, restriction enzymes) to detect target sequences Post-transduction removal of ITRs resulted in multiple advantages.
rather than using PCR amplicons. As such, nuclease-mediated loss of First, the inter-cluster polydispersity was decreased after intracellular
linkage is the best choice for genotyping and quantifying concatemeric removal of the ITRs. Previous research suggests that the ITR primes the
knockins shortly after adding AAV or in regions that are difficult to single-stranded AAV vector genome for second-strand synthesis28. It
amplify by PCR. is only after second-strand synthesis occurs that the vector genes are
Although the AAV genome is known to form linked episomal seg- expressed. Because MOI and second-strand synthesis may vary cell to
ments25, conventional analytical methods are unsuited to detecting cell, vector genes are expressed at varying levels leading to interclus-
them for several reasons. First, methods must be designed to consider ter polydispersity. This fades after the vector DNA is diluted through
detection of concatemeric insertions. In–out PCR, a more common numerous cell divisions. Because the DC method decreases the polydis-
genotyping method, will not detect concatemers. Second, concate- persity, it is possible that Cas9 removes the ITR before second-strand
meric knockins are easiest to detect in subclones. This is because the synthesis occurs, leaving a long single-stranded DNA template. This is

Nature Biotechnology
Article
possible because Cas9 has been shown to cut single-stranded DNA29.
In contrast, the IC method may cut the ITR in a hairpin, resulting in
a double-stranded break; however, we did not detect an increase in
NHEJ-mediated knockins. Regardless, the DC method may have more
appeal because only a single RNP is used, minimizing the chance of
additional off-target cuts in the genome. In all cases, NC, IC and DC
methods have similar concentrations of episomal vector DNA within
the cell 5 d after editing. This suggests that the decreased polydisper-
sity after ITR removal is due to decreased episomal expression, which
ultimately leads to more defined clusters and fewer false positives. The
second advantage of ITR removal is the clear decrease in concatemeric
knockins. Because these concatemers can create unexpected problems
(Fig. 5), their prevention is critical to ensuring reproducible, safe and
high-quality gene modifications. However, ITR removal also resulted
in a decrease in double-positive cells when performing dual-color
knockins. This is not surprising because cells in the double-positive
population also have the highest rate of concatemeric knockins. The
decrease in editing efficiency is minor compared to the increase in
bona fide knockins.
Gene-modified HSPCs can be transplanted to cure genetic dis-
eases. Here we show that our methods can be immediately applied to
existing pipelines to decrease the concatemer rate. Engraftment rates
suggest that ITR removal does not negatively affect HSC function and
may even enhance engraftment. The potential increase in engraftment
efficiency could be due to reported genotoxic effects of the ITR30, which
may be mitigated in the IC and DC methods. However, the disposition
of the ITR fragment after cleavage is unclear, and more work is needed
to analyze the effect of ITR removal on HSC function.
Although we did not reach a 0% concatemer rate, a 10-fold decrease
is an important first step. Future experiments could modify the ITR or
modulate DNA repair pathways to further decrease concatemeric
rate. Regardless, having available tools to properly assess a genotype
is critical, particularly when selecting a clonally expanded population
of edited cells or when developing safety profiles.
Online content
Any methods, additional references, Nature Portfolio reporting sum-
maries, source data, extended data, supplementary information,
acknowledgements, peer review information; details of author contri-
butions and competing interests; and statements of data and code avail-
ability are available at https://doi.org/10.1038/s41587-024-02171-w.
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Article
Methods
Ethical use of stem cells
All experiments were performed in accordance with ethical recommen-
dations put forth by the International Society for Stem Cell Research
in the 2021 Guidelines for Stem Cell Research and Clinical Translation.
Oversight was conducted and approved by Stanford University’s
Research Compliance Office and the corresponding Stem Cell Research
Oversight panel.
PSC lines and maintenance
Four human PSC lines were used in this study. Patient-derived iPSCs
(PT-iPSC, iSU223n)31,32, H9 embryonic stem cells (ESC, WiCell), H1 ESCs
(WiCell) and wild-type iPSCs (WT-iPSC, SUN004.1)33 and their derivative
subclones were routinely propagated feeder free in StemFit Basic02
medium supplemented with 20 ng ml−1 bFGF or Basic04 complete type
(Ajinomoto) on cell culture plastics with iMatrix-511 (Nippi) basement
membrane matrix, with single-cell dissociation by using TrypLE Express
Enzyme (Gibco). Y-27632 dihydrochloride (Tocris) was added for 24 h
after passage unless otherwise indicated. PT-iPSCs were used in Figs. 1
and 2 and Extended Data Figs. 1–4 and 7. In all other cases, specific PSC
lines are indicated in the text and figures.
AAV vector production
AAV vector plasmids were cloned in the pAAV-MCS plasmid containing
ITRs from AAV serotype 2 (AAV2)19. CD14 vector contained P2A and Cre.
AAVS1 vectors contained a UBC promoter and CreERT or fluorescence
reporter genes, such as GFP or mCherry. HBB, TET2, RUNX1 and IL2RG
vectors contained UBC promoters and fluorescence reporter genes,
such as GFP or mCherry. The homology arms for CD14, AAVS1, TET2 and
IL2RG were approximately 400 bp (379–414 bp), and the left and right
homology arms for HBB were 537 bp and 420 bp, respectively. Vectors
with DC sites for ITR removal contained the same sequences as genomic
sgRNA target sequences, with the external PAM (ExP) facing inward
between the ITR and homology arm. AAV6 particles were produced
in 293FT cells (Thermo Fisher Scientific) transfected using standard
PEI transfection with ITR-containing plasmids and pDGM6 containing
the AAV6 cap genes, AAV2 rep genes and adenovirus serotype 5 helper
genes. Particles were harvested after 72 h, purified using the AAVpro
Purification Kit (Takara Bio) according to the manufacturerʼs instruc-
tions and then stored at −80 °C until further use. Viral vector titers
measured as vector genomes per microliter (vg/µl) were determined
by ddPCR analysis of serially diluted vector samples using primers and
probes that target the vector ITR (Supplementary Table 1).
Electroporation and transduction of cells
All synthetic sgRNAs and Cas9 protein were purchased from Inte-
grated DNA Technologies. The genomic sgRNA target sequences
with PAM were: HBB, 5′-CTTGCCCCACAGGGCAGTAACGG-3′;
IL2RG, 5′-TGGTAATGATGGCTTCAACATGG-3′; RUNX1, 5′-TACCTT
GAAAGCGATGGGCAGGG-3′; AAVS1, 5′-GGGGCCACTAGGGACAG
GATTGG-3′; TET2, 5′-TCATGGAGCATGTACTACAATGG-3′; and CD14,
5′-CTAGCGCTCCGAGATGCATGTGG-3′.
Cas9 RNP was made by incubating protein with sgRNA at a
molar ratio of 1:2.5 at 25 °C for 10 min immediately before elec-
troporation into CD34+ HSPCs or iPSCs/ESCs, with a final concentra-
tion of Cas9 at 150 ng µl−1 during electroporation. For experiments
employing the IC method of ITR removal, sgRNA of ITR sequence
(5′-GCGCGCTCGCTCGCTCACTGAGG-3′) was separately incubated
with Cas9 and added at the same final concentration in addition to the
other RNP. Both CD34+ HSPCs and iPSCs/ESCs were electroporated
using the Lonza Nucleofector 4D (program DZ-100 for CD34+ HSPCs
and program CB-150 for iPSCs/ESCs). Immediately after electropo-
ration, AAV6 donor vectors were added at an MOI (vector genomes
per cell) of 10,000 for CD34+ HSPCs, 5,000 for PT-iPSCs and 1,000
for WT-iPSCs/ESCs, unless indicated otherwise in the figures. After
Nature Biotechnology
https://doi.org/10.1038/s41587-024-02171-w
overnight incubation, cells were washed. For iPSCs/ESCs, Y-27632
dihydrochloride was added to media 48 h after electroporation.
Flow cytometry and subcloning
Cells were washed and stained with propidium iodide at a final concen-
tration of 1 μg ml−1 or DAPI at a final concentration of 0.1 μg ml−1 right
before analysis or sorting. Cell analysis and cell sorting were performed
on a FACSAria II (BD Biosciences). To subclone edited PSCs, 5 d after
electroporation, DAPI-negative and fluorescence-positive cells ( just
DAPI-negative cells for no-marker vector in CD14 P2A-Cre and AAVS1
UBC-CreERT2) were sorted into 96-well plates. After 7 d, the subclones
were further expanded in 12-well plates. Edited PSCs that were not sub-
cloned (data in Fig. 3g and Extended Data Figs. 6 and 7f) were sorted
5 d after electroporation into 12-well plates, followed by a second sort
approximately 2 weeks later to ensure purity. For edited CD34+ cells,
3 d after electroporation, DAPI-negative and fluorescence-positive cells
were sorted into 150 μl of myeloid differentiation media consisting of
MyeloCult H5100 (STEMCELL Technologies) supplemented with SCF
(20 ng ml−1), TPO (20 ng ml−1), Flt3-Ligand (20 ng ml−1), IL-6 (20 ng ml−1),
IL-3 (20 ng ml−1), GM-CSF (20 ng ml−1) and G-CSF (20 ng ml−1). Approxi-
mately 3 weeks later, large fluorescent colonies were used for subse-
quent analysis.
DNA extraction
For ddPCR, qPCR and standard PCR analysis, DNA was extracted using a
crude lysis buffer containing 0.1% SDS, 5 mM EDTA, 0.2 mg ml−1 protein-
ase K (Thermo Fisher Scientific), 15 mM tris (pH 8.2) and 100–200 mM
NaCl. For PSCs, 100 µl of lysis buffer was added to approximately 1 × 105
to 1 × 106 pelleted cells, followed by incubation at 55 °C for 10 min and
heat inactivation at 80 °C for 10 min. For HSPCs, 35 µl of lysis buffer
was added to approximately 5 × 103 to 1 × 105 cells, followed by the same
heating protocol. All samples were homogenized by pippette tritura-
tion until smooth, and debris was pelleted through centrifugation at
6,000g for 3 min.
For Southern blots, genomic DNA was purified from approxi-
mately 1 × 107 cells using multiple columns from the DNeasy Blood
and Tissue Kit (Qiagen) and following the recommended protocol
with RNase A.
CB processing
Frozen mononuclear cells derived from cord blood (CB) were pur-
chased from the New York Blood Center. CD34+ HSPCs were isolated
using a human CD34 MicroBead Kit (Miltenyi Biotec). CD34+ HSPCs
were cultured in HSPC expansion media consisting of StemSpan SFEM
II (STEMCELL Technologies) supplemented with SCF (20 ng ml−1),
TPO (20 ngm l−1), Flt3-Ligand (20 ng ml−1), IL-6 (20 ng ml−1) and UM171
(35 nM). Cells were cultured at 37 °C, 5% CO2 and 5% O2.
HSPC transplantation
Three days after electroporation/transduction, GFP+ edited CD34+ cells
were sorted (2 × 105 to 3 × 105 cells) and transplanted into one femur
of sub-lethally irradiated mice (200 rad, 2–24 h before transplant).
Four months after transplantation, the human cell-engrafted mice
were euthanized, and all bone marrow was harvested by crushing
the bones. Non-specific antibody binding was blocked (human and
mouse Fc block, BD Biosciences) and stained (30 min, 4 °C, dark) with
PE-Cy7-conjugated anti-mouse CD45.1 antibody (A20, Invitrogen),
PE-Cy5-conjugated anti-mouse TER-119 antibody (TER-119, Invitrogen),
V450-conjugated anti-human CD45 antibody (HI30, BD Horizon) and
PE-conjugated anti-HLA-ABC antibody (W6/32, eBioscience) and ana-
lyzed by flow cytometry. For cell sorting, the engrafted human cells were
isolated using human CD45 MicroBeads (Miltenyi Biotec). The enriched
human cells were stained (30 min, 4 °C, dark) with V450-conjugated
anti-human CD45 antibody and PE-conjugated anti-HLA-ABC antibody,
isolated by flow cytometry and analyzed by digital PCR. Antibodies
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were diluted in accordance with the manufacturerʼs recommendations
with fewer than 1 × 106 cells per 50 µl.
Mice and animal care
All mouse experiments were conducted according to a protocol
approved by the Institutional Animal Care and Use Committee (Stanford
Administrative Panel on Laboratory Animal Care, no. 22264) and
adhering to the National Institutes of Health’s Guide for the Care and
Use of Laboratory Animals. Six- to 8-week-old male or female NOD.
Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were used (The Jackson Labo-
ratory). All mice were housed in a pathogen-free animal facility in microi-
solator cages, and the experimental protocol was approved by Stanford
University’s Administrative Panel on Lab Animal Care (no. 22264).
Southern blot genotyping
Southern blot analysis was performed to genotype the CD14 locus after
knocking in CRE in PT-iPSCs. Standard methods for Southern blot with
genomic DNA were applied. In brief, 10 µg of each DNA sample was
subjected to restriction digest with 200 units of NdeI (New England
Biolabs) and 200 units of BlpI (New England Biolabs). Swiss-Webster
albino mouse genomic DNA (Promega) was used as a negative control.
For preparation of size standards, 6.6 × 106 copies of a 22-kb plasmid
that contained the right homology arm CD14 sequence was spiked into
1.5 µg of mouse gDNA and digested in a volume of 100 µl with various
combinations of enzymes to yield the desired fragment size. A total
of seven different size standard digests were performed separately
and pooled only after addition of phenol-chloroform-isoamyl alcohol
(Invitrogen). Fragments were phenol-chloroform purified following
the overnight restriction digest and precipitated using one volume of
2-propanol after the addition of 1/10 volume of 3 M sodium acetate and
5 µl of GlycoBlue (15 mg ml−1, Invitrogen). After an overnight incubation
at −20 °C, fragments were pelleted by centrifugation at 4 °C, washed
with 70% ethanol, air dried and resuspended in 40 µl of Tris-EDTA buffer
with gel loading buffer (Blue Juice, Invitrogen). Samples were loaded
onto a 10 × 6-inch 0.8% agarose gel using TAE as running buffer. The
gel was run at 28 V for 24 h, incubated with denaturating buffer (3 M
NaCl, 0.4 M NaOH) twice for 30 min with agitation and incubated in
transfer buffer (3 M NaCl, 8 mM EDTA) for 15 min with agitation. The gel
was then blotted overnight onto a positively charged nylon membrane
(Roche) using a piece of Whatman paper serving as a wick to transfer
the buffer and, thus, the DNA from the gel onto the membrane by the
means of capillary action. After crosslinking (GS GeneLinker, Bio-Rad),
the membrane was pre-hybridized with 10 µg ml−1 salmon sperm DNA
in PerfectHyb Plus Hybridization buffer (Sigma-Aldrich) for a minimum
of 2 h at 60 °C with rotation. The probe was generated by amplification
of the 400-bp-long right homology arm sequence from the rAAV vector
using primers RHA-F (5′-GCGTGGTCCCAGCCTGTGC-3′) and RHA-R
(5′-GCAGCCCTAGCCAGGAGTC-3′). This fragment encompasses part
of the CD14 coding sequence, the 3′ UTR and some intergenic sequence
downstream of the CD14 gene. The amplicon was gel purified, and 10 ng
was labeled with [a-32P] dCTP using the BcaBEST Labeling Kit (Takara
Bio) according to the manufacturer’s instructions. Unincorporated
nucleotides were removed with an Illustra MicroSpin G-25 column, and
the probe was added to the pre-hybridized membrane. Hybridization
was allowed to occur for 2–3 d at 60 °C with rotation. The membrane
was washed twice under low-stringent conditions (2× SSC, 20 min at
room temperature), followed by one wash under high-stringent con-
ditions (2× SSC with 0.1% SDS, 30 min at 60 °C). The membrane was
exposed onto a phosphoimager screen and visualized using a Personal
Molecular Imager (Bio-Rad). Band sizes were determined by interpola-
tion between the log of adjacent size standards.
PCR genotyping
Two types of PCR genotyping strategies were used: three-primer in–out
PCR24 and two-primer PCR. Approximate primer locations are shown in
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Fig. 1b. Three-primer in–out PCR can be used to analyze either the left
side (PCR-L) or the right side (PCR-R) of the knockin site. Two-primer
PCR spans the full knockin site (PCR-F). Both methods result in an
expected banding pattern shown in Fig. 1c. SeqAmp DNA Polymerase
(Takara Bio) was used for PCR amplification. All PCR reactions were
run following the manufacturerʼs recommendations at a final volume
of 20 µl with 0.5–1 µl of extracted DNA and 0.25 µM of each primer. All
reactions were loaded on a 1% agarose gel in TAE mixed with 1:10,000
SYBR Safe (APExBio). Two microliters of TrackIt 1 Kb Plus (Invitrogen)
or GeneRuler 1 kb (Thermo Fisher Scientific) was used for ladders. PCR
product was diluted 1:6 with loading buffer, and 5–10 µl was loaded
in each lane. In–out PCR was also used to analyze GFP and mCherry
knockins at the TET2 locus in Extended Data Fig. 7. In this case, only
two primers were used for PCR-L and PCR-R because all samples had a
biallelic concatemer-free genotype. All primer sequences are listed in
Supplementary Table 1.
Junction-spanning PCR and sequencing
PCR was used to amplify a product spanning the concatemer junction
as shown in Extended Data Fig. 3a using similar PCR conditions
as described above. Samples were the same as those analyzed by
Southern blot. Then, 10 µl of PCR product was analyzed by gel elec-
trophoresis. For sequencing, upper and lower bands were cut from
the gel and purified with a NucleoSpin Gel and PCR Clean-Up Kit
(Machery-Nagel). The bands were submitted for Sanger sequencing
(Elim Biopharmaceuticals) and Primordium Labs’ nanopore-based
sequencing (Primordium).
qPCR
ITR qPCR was performed on a QuantStudio 7 Flex Real-Time PCR System
(Applied Biosystems) using TaqMan Fast Advanced Master Mix (Applied
Biosystems); final reaction volume was 10 µl in a 384-well plate. All
samples were analyzed with ITR and CD14-LHA primer/probe sets
(listed in Supplementary Table 1) at a final concentration of 0.45 µM
and 0.125 µM for primers and probes, respectively. The analyzed human
genomic DNA was extracted from PT-iPSCs with CRE knocked in to CD14
(sample 10 and sample 14 in Extended Data Fig. 1). Samples were diluted
to similar molar concentrations (final concentration of ~300 copies of
CD14-LHA per microliter as determined by ddPCR without restriction
enzymes). Both samples were analyzed with and without 2.5 units of
the restriction enzyme AhdI (New England Biolabs) added directly to
the qPCR mixture. Thermocycler conditions were run in accordance
with the manufacturerʼs recommendations with the addition of an
initial 37 °C × 5-min incubation.
Microscopy
Cells were imaged using the Operetta High Content Imaging System
(PerkinElmer) with GFP and mCherry fluorescent filter sets. Harmony
version 3.5.2 (PerkinElmer) was used to export microscopy data. ImageJ
1.52p (National Institutes of Health) was used to subtract background
and measure the average GFP and mCherry fluorescent intensity within
the cell colonies. In Extended Data Fig. 4c, images are displayed using
individualized or uniform imaging processing. For individualized
image processing, auto-brightness-&-contrast was used to set display
thresholds before converting and combining images. For uniform pro-
cessing, display thresholds were set to the same values for all images.
Uniform processing was also used in Fig. 2h.
Polydispersity in flow cytometry quadrants
Polydispersity in flow cytometry plots was measured as relative change
in average absolute deviations. In brief, the four centroids of the cell
cluster in each quadrant were calculated separately, using GFP as the
x axis and mCherry as the y axis. The average distance of each cell to
the respective centroid was then measured, and IC and DC measure-
ments were normalized to the control (NC). This was repeated for the
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seven groups shown in Extended Data Fig. 5a–g, and the average of all
groups was plotted in Extended Data Fig. 5h. All measurements were
performed with linear (not log-transformed) data points.
Digital PCR genotyping: concept
Three methods of digital PCR genotyping were developed. Each
method can detect the basic genotype (that is, wild-type, monoallelic
and biallelic) in subcloned cells, whereas the methods have varying
capabilities to detect other more complex genotypes, such as concate-
meric knockins. All assays use relatively short amplicons (<150 bp),
which allows efficient multiplexing and shorter run times. Up to four
primer/probe sets are multiplexed per reaction. Because only two
channels are used (FAM and HEX), amplitude multiplexing34 is neces-
sary and results in two-dimensional plots, as shown in Extended Data
Fig. 1g. Samples are often analyzed multiple times with various restric-
tion enzymes added directly to the ddPCR reaction. These restriction
enzymes are necessary and play direct roles in determining complex
genotypes, as discussed below.
Allele counting. This genotyping strategy is a copy number variation
(CNV) analysis, which measures the concentration of various genetic
segments and compares their ratios to determine copies per cell and
corresponding genotype. Four primer/probe sets are multiplexed
(Extended Data Fig. 1f):
• Ref1 and Ref2: The first two primer/probe sets target two differ-
ent reference sites. These serve both as a reference for overall
cell number and a quantitation control. Concentrations of the
two references should always measure at a 2:2 ratio (2 is used
because most reference genes exist twice per cell). If the ratio is
off by more than 10%, there may be problems, such as abnormal
karyotypes, DNA degradation or improper gating, and repeats
should be considered. The two references can be in cis or in trans
with the knockin site.
• No_KI: The third primer/probe set is designed to span the
genomic cut site (that is, knockin site), such that the primers and
probe are at least 25 bp from the cut. This primer/probe set will
detect the wild-type allele or alleles with indels, but it will fail if
there is a large knockin due to the increased distance between
the forward and reverse primers. This primer/probe set is used
to determine if a knockin is wild-type, monoallelic or biallelic.
It is important to ensure that larger Cas9-induced deletions
(>25 bp) are infrequent at the measured loci before employing
this method.
• KI: The fourth primer/probe set is designed to target the knockin
sequence, homology arm or any other part of the vector genome
depending on the purpose. This primer/probe set can be used
to determine if a knockin is concatemeric, off-target or NHEJ
mediated.
The concentration of each target and corresponding copies per
cell are calculated as shown below.
C = (ln(ntotal ) − ln(nneg ))/0.00085
C’ = C/(((CRef 1 /aRef 1 ) + (CRef2 /aRef2 ))/2)
C: volumetric concentration (copies/µl)
C’: cellular concentration (copies/cell)
ntotal: total number of ddPCR droplets
nneg: number of ddPCR droplets that are negative for a given
measured target
a: alleles per cell for a given locus (usually 1 or 2)
The constant 0.00085 indicates the average droplet volume in
microliters used in our ddPCR equipment. a = 2 for most experiments.
a = 1 for reactions that use IL2RG references in male cells.
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To determine the total number of insertions after performing a
knockin, this assay must be run with restriction enzymes that separate
concatemeric knockins (Extended Data Fig. 1j). When using a primer/
probe set to target the KI gene segment, extra insertions are indicated
by copies per cell greater than 0, 1 or 2 for wild-type, monoallelic or bial-
lelic genotypes, respectively. However, if reanalyzed without restriction
enzymes, the number of measured insertions per cell should decrease
if concatemeric insertions exist (Extended Data Fig. 1k). This assay can
be used in this way to measure the number of concatemeric inserts per
cell in subcloned samples.
Compared to the linkage analysis assay discussed below, the
allele counting assay has two advantages. First, the linkage analysis
assay requires at least two runs (that is, with and without restriction
enzymes) to determine genotype and the total number of insertions.
In contrast, the allele counting assay requires only a single run (that
is, with restriction enzyme); thus, it works well as a quick screen to
select subcloned cell lines with intended genotypes. Second, the
references designed for this assay work in trans and can be located
anywhere on the genome. Therefore, they can be recycled for other
assays.
Linkage analysis. This strategy compares the linkage of two or more
gene segments to determine genotypes. Linkage can be determined by
assessing the number of digital PCR droplets that are positive for two
or more gene segments targeted by different primer/probe sets. Three
to four primer/probe sets are multiplexed (Extended Data Fig. 4a):
• Ref1 and Ref2: The first two primer/probe sets target two dif-
ferent cis reference sites that flank both sides of the intended
genomic knockin site. These primer/probe sets amplify short
regions located 0–1,000 bp outside the homology arms and
should be linked if DNA degradation has not occurred. Similar
to the allele counting strategy described above, these primer/
probe sets serve as references for overall cell numbers and
quantitation controls. Because these primer/probe sets are in cis
and are nearby the editing site, these references also serve two
additional purposes. First, digital PCR linkage analysis can be
used to determine if these references are linked to knockin gene
segments to determine genotypes. Second, the two references
serve as linkage controls to ensure that the DNA is not consider-
ably fragmented or degraded after purification, which would
diminish the measured linkage of nearby gene segments. We
find that this works best if the two references have more than
75% linkage.
• KI: Another primer/probe set is designed to target knockin
sequences or other parts of the vector genome, depending on
the purpose. Linkage analysis can be used to determine the
percentage of references that are linked to KI and infer complex
genotypes.
Linkage analyses are not bidirectionally equivalent. For example,
in the case of a monoallelic knockin of GFP into a human autosome,
100% of GFP will be linked to Ref1 (written as GFP → Ref1 = 100%), but
only 50% of Ref1 will be linked to GFP (written as Ref1 → GFP = 50%).
Additionally, because Ref2 → GFP should be similar to Ref1 → GFP, the
average is usually used to determine Ref → GFP.
To determine the basic genotype, this assay must not be run with
restriction enzymes that cut within the knockin sequence or homol-
ogy arms. Restriction enzymes that cut outside of the reference sites
are acceptable, and they may be necessary to homogenize DNA and
minimize digital PCR rain. However, this assay should be run a second
time for all samples with restriction enzymes that separate potential
concatemers to determine the total number of insertions as deter-
mined by allele counting CNV (Extended Data Fig. 4b).
The calculation for determining the percent linkage of
Targ1 → Targ2 is:
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%LTarg1→Targ2 = (1− ((CTarg1_or_Targ2 −CTarg2 ) /CTarg1 ) ) ∗ 100
%L: percent linkage
C: volumetric concentration (copies/µl)
CTarg1_or_Targ2: determined by treating any droplet positive for Targ1
and/or Targ2 as positive
Linkage analysis has multiple advantages over allele counting.
First, it is easier to design because there is more latitude for the cis
reference primer/probe sets than there is for the No_KI primer/probe
sets. Second, because the No_KI primer/probe set is not needed, it can
be replaced by another primer/probe set. This is particularly useful
when analyzing double knockins, such as GFP and mCherry, because
both targets and references can be multiplexed in one reaction. Third,
large deletions that would disrupt the No_KI primer/probe set would
be less disruptive because the references are located hundreds of
base pairs away. Fourth, episomal DNA or off-target insertions can be
distinguished from on-target insertions by measuring linkage between
cis references and insert sequences. This is a unique property of this
assay and is particularly useful if long-range PCR and Southern blots
are not practical.
Linkage analysis can also be used to detect ITRs or other viral
vector regions linked to genomic loci, enabling the quantification
of %-alleles-with-concatemeric-KI. Therefore, linkage analysis can
measure the allelic abundance of concatemers in bulk edited samples
without subcloning. This would not work well with ITR CNV because
the CNV alone does not distinguish between on-target and off-target
knockins. Finally, when coupled with restriction enzymes that cut only
one side of inserts, linkage analysis can determine orientations and/or
sequences of multiple knockins.
Nuclease-mediated loss of linkage. This strategy employs nucleases
(that is, restriction enzymes, Cas9, etc.) to recognize and quantify gene
segments. Cis reference primer/probe sets are designed for linkage
analysis as described above. The ddPCR reaction is then run with and
without nucleases that target unique sites within knockins or other
parts of the vector genome. If the recognition site exists between the
reference primer/probe sets, linkage between the two reference sites
will be lost. This can be quantified by the equation below:
%allelesTarget_KI = (1− (%Lwith_nuclease /%Lwithout_nuclease ) ) ∗ 100
This strategy has three unique advantages. First, only two primer/
probe sets are used, lowering cost and complexity. Instead of KI or
No_KI primer/probe sets, nucleases such as restriction enzymes are
used, which many laboratories readily have. Second, PCR-resistant
sequences, such as sequences with inhibiting secondary structures, do
not have to be efficiently amplified by primer/probe sets to be detected.
Third, the assay is agnostic to episomal vector DNA and can, therefore,
detect knockins or concatemers shortly after editing without waiting
for episomal DNA to dissipate. Although linkage analysis can also distin-
guish between episomal and on-target sequences, ddPCR reactions can
easily be overloaded and pushed outside of their dynamic range when
too many episomal copies of DNA exist, such as shortly after editing.
Digital PCR genotyping: procedure
Each ddPCR reaction was prepared and analyzed with a QX200
ddPCR system (Bio-Rad) and ddPCR Supermix for Probes (No dUTP)
per Bio-Rad’s standard recommendations, unless otherwise stated.
QuantaSoft version 1.6.6.0320 (Bio-Rad) was used to operate the
machine. All reactions were mixed to 25 µl and contained up to 1–5 µl
of DNA. Then, 2.5–5 U of restriction enzyme was frequently added,
and digestion was performed immediately before droplet generation
at 37 °C × 5 min. For droplet generation, 20 µl was loaded into the
droplet generator cassette in groups of eight per Bio-Rad’s protocol.
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Thermocycler conditions: 95 °C × 10 min; 50 cycles of 94 °C × 30 s and
60 °C × 60 s; 98 °C × 10 min; and hold at 4 °C. Cluster identification
was performed manually with QuantaSoft Analysis Pro version 1.0.596
(Bio-Rad). Cluster information was exported into Microsoft Excel for
further analysis. Forward primer, reverse primer and probe were at a
3.6:3.6:1 ratio. Primer and probe sequences are listed in Supplementary
Table 1. Restriction enzymes and multiplexing parameters are listed in
Supplementary Table 2. Raw digital PCR cluster information is listed in
Supplementary Table 3. NC/IC/DC PSC subclone genotypes and NC/DC
HSPC subclone genotypes are summarized in Supplementary Table 4.
Terminology:
• Insert: DNA sequence corresponding to a part of the vector
genome (for example, CRE, GFP, mCherry, etc.); the number of
measured insertions may change if analyzed with and without
restriction enzymes that separate concatemers
• KI: an allele at the target locus (for example, CD14, AAVS1, etc.)
that contains at least one insert
• No KI: an allele at the target locus that does not contain any
inserts
• On-target insert: insert that is knocked into target locus
• Off-target insert: insert that exists in the cell but not at the target
site
• Extra insert: number of insertions greater than 1 or 2 for monoal-
lelic and biallelic knockins, respectively
• Concatemeric insert: extra on-target inserts
• +/−RE: indicates an analysis is performed with (+) or without
(−) restriction enzymes (New England Biolabs) that cut sites
to separate concatemers (some −RE runs may have restriction
enzymes added to enhance ddPCR; however, they will not cut
any sequence within the editing site nor any sequence flanked by
cis references)
To analyze genotype after knocking in CRE at the CD14 locus in
human iPSCs (results shown in Fig. 1e,g and Extended Data Fig. 1h,i),
the ddPCR allele counting strategy was used. Two trans references were
multiplexed with a CD14_No_KI primer/probe set and a CRE primer/
probe set. The reaction was run with and without a restriction enzyme
that separates concatemers. C′CD14_No_KI rounding to 0, 1 or 2 was scored
as biallelic, monoallelic and wild-type, respectively. Extra insertions
were determined by the equation:
C’Extra_CRE = C’CRE_+RE − (2 − C’CD14_No_KI )
Similar primers/probes were used to analyze genotypes after
knocking in Ubc-CRE at the AAVS1 locus in human iPSCs (results shown
in Fig. 1h and Extended Data Fig. 2b,c). However, the AAVS1_No_KI reac-
tion did not run efficiently and required addition of the BspEI restric-
tion enzyme. This enzyme does not cut within the editing site, so it was
added to all reactions using this primer/probe set to eliminate rain. The
reaction was also run with and without another restriction enzyme
that separates concatemers. Genotyping was performed similarly to
the CD14 locus.
To count the number of LHA and RHA gene segments after knock-
ing in CRE at the CD14 locus in human iPSCs (results shown in Fig. 2d
and Extended Data Fig. 3e,f), the ddPCR allele counting strategy was
used. Two CD14 cis references were multiplexed with primer/probe sets
targeting LHA and RHA. The reaction was run +/−RE. Extra LHA and RHA
insertions were accounted for by subtracting 2 from their measured
copies per cell in the +RE reaction. Vector ITRs inserted into the genome
were also quantified. These reactions were run with the same two CD14
cis references, a CRE primer/probe set and a primer/probe set targeting
the vector ITR region. The reaction was run three times (with no RE, with
AhdI and with MseI and AhdI) and analyzed by allele counting (Fig. 2d
and Extended Data Fig. 3i). To determine if concatemeric knockins
occurred in one or two alleles, the dataset was analyzed by linkage
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analysis. Specifically, CD14_Ref1 → ITR and CD14_Ref2 → ITR were
measured on the samples run with AhdI (Extended Data Fig. 3k, upper
and middle row). Genomic ITR insertions were further validated with
nucleus-mediated loss of linkage, by analyzing CD14_Ref1 → CD14_Ref2
on the same +AhdI dataset (Extended Data Fig. 3k, bottom row). This
method showed that sample 14 had at least one ITR inserted into the
target site, but its orientation (that is, lack of linkage to references when
adding AhdI) suggests that it was an NHEJ-mediated insertion. This is
further supported by allele counting, which shows that the sample has
an additional LHA and RHA but only one copy of CRE.
All subclones (both PSCs and HSPCs) with knockins of
Ubc-mCherry and/or Ubc-GFP were genotyped by ddPCR linkage
analysis. Each loci had two cis references designed as described above,
which were multiplexed with Ubc-mCherry and Ubc-GFP primer/probe
sets. Each reaction was run with and without restriction enzymes that
separate concatemeric knockins. For basic genotyping calculations,
linkage analysis was performed by pooling Ubc-mCherry and Ubc-GFP
clusters into a single ‘insert’ population. Because linkage analysis is
unidirectional, the average linkages were determined by:
%LAvg_Ref→Ref = (%LRef 1→Ref2 + %LRef2→Ref 1 )/2
%LAvg_Ref→Ins = (%LRef 1→Ins + %LRef2→Ins )/2
A correction can be made for DNA degradation:
%LCorr_Avg_Ref→Ins = %LAvg_Ref→Ins /%LAvg_Ref→Ref ∗ 100
KI and No KI alleles were calculated from the digital PCR run −RE:
C’KI =%LCorr_Avg_Ref→Ins ∗a/100
C’No_KI = a − C’KI
Basic genotypes were determined by rounding C′No_KI to the nearest
integer. 0, 1 and 2 indicate biallelic, monoallelic and wild-type geno-
types, respectively.
All runs were reanalyzed with +RE, and the total copies per cell
of GFP and mCherry were calculated as described in allele counting.
Runs that did not have the following quality parameters were excluded:
total droplet number >8,000; the concentration of Ref1 is no more
than 10% different than the concentration of Ref2; the average concen-
tration of Ref1 and Ref2 is >12 copies per microliter; %LAvg_Ref→Ref >75%
without fragmenting restriction enzyme and <15% with fragmenting
restriction enzyme; and the copies per microliter of GFP and mCherry
without fragmentation is not >20% the copies per microliter of GFP and
mCherry with restriction enzyme.
If both runs (+/−RE) passed quality control, more complex geno-
types were determined. Off-target and concatemeric insertions were
determined by rounding each value in the following equations to the
nearest integer:
C’Off−targ_Ins = C’Ins_−RE − C’KI
C’Concat_Ins = C’Ins_+RE − C’KI − C’Off−targ_Ins
Linkage between GFP and mCherry was determined to be true if the
sum of %LGFP→mCh and %LmCh→GFP was more than 20% when analyzed −RE.
For bulk genotyping, Ubc-GFP was knocked in to the target locus,
and DNA was extracted from a group of GFP+ cells for analysis. This
is distinct from previous genotyping analyses, which had all been
performed on DNA extracted from a pure, clonal population. Bulk
genotyping was performed on the vector dilution series in PSCs
(Fig. 3g and Extended Data Fig. 6), and the human cells recovered
after HSPC xenotransplantation (Fig. 4e). For these experiments, the
vector backbone was modified such that two unique DNA sequences
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less than 100 bp were inserted flanking the homology arms, as shown
in Extended Data Fig. 6a. Primer/probes sets were designed to detect
these two sequences, termed ID1 and ID2. The bulk samples were first
analyzed −RE with cis reference primer/probe sets and the GFP primer/
probe set and then reanalyzed with the No_KI primer/probe set. These
datasets were used to calculate percent KI alleles using both linkage
analysis and allele counting:
%KI = %LCorr_Avg_Ref→GFP
%KI = (1 − (CNo_KI / ((CRef 1 + CRef2 )/2)) ) ∗ 100
To detect on-target concatemeric knockins, the samples were
analyzed −RE with the cis references and ID primer/probe sets. The
percent of KI alleles with concatemeric insertions was determined by:
%LAvg_Ref→ID = (%LRef 1→ID1_or_ID2 + %LRef2→ID1_or_ID2 ) /2
%LCorr_Avg_Ref→ID = (%LAvg_Ref→ID /%LAvg_Ref→Ref ) ∗ 100
%KIConcat = (%LCorr_Avg_Ref→ID /%KI) ∗ 100
To determine the average size of concatemers, the samples were
run with the cis references and GFP primer/probe sets +RE. The average
concatemer size (including the first insert) was calculated by:
Avg_concat_size = ((C’GFP_+RE − C’GFP_−RE )/(%KIConcat /100))+1
Statistics and graphs
ddPCR one-dimensional and two-dimensional plots were generated
with QuantaSoft Analysis Pro version 1.0.596 (Bio-Rad). Flow cytom-
etry graphs were generated with FlowJo version 10.8 (BD Biosciences).
All other graphs were generated in Microsoft Excel and PowerPoint.
Linear regressions were measured in Microsoft Excel. Significance
was calculated with two-sided t-tests using Microsoft Excel. For paired
t-tests, the following function was used:
p-value = T.TEST(array1,array2,2,1)
For unpaired t-tests, the following function was used:
p-value = T.TEST(array1,array2,2,2)
Reporting summary
Further information on research design is available in the Nature
Portfolio Reporting Summary linked to this article.
Data availability
All data generated or analyzed during this study are included in the
published article and its supplementary information files. Regarding
digital PCR data, raw individual cluster information is included in Sup-
plementary Table 3 in accordance with the 2020 guidelines regarding
the minimum information necessary for publication of quantitative
digital PCR experiments.
References
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switch in human pluripotent stem cells and disease cells. Gene
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32. Chao, M. P. et al. Human AML-iPSCs reacquire leukemic properties
after differentiation and model clonal variation of disease. Cell
Stem Cell 20, 329–344 (2017).
33. Ang, L. T. et al. Generating human artery and vein cells from
pluripotent stem cells highlights the arterial tropism of Nipah and
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Article
34. Whale, A. S., Huggett, J. F. & Tzonev, S. Fundamentals of
multiplexing with digital PCR. Biomol. Detect. Quantif. 10,
15–23 (2016).
Acknowledgements
We thank K. C. Chan, M. Rivera, F. Zhao and S. Homma for laboratory
and administrative support. We thank S. Tsuji for scientific advice.
This work was supported by grants from the National Institutes of
Health (NIH) (R01DK121851, H.N.; R21OD030009, H.N.; R21OD030529,
H.N. and R.M.; and R01HL064274, M.A.K.). F.P.S. received mentorship
and financial support from Stanford’s SPARK Translational Research
Program; the Stanford Clinical and Translation Science Award
to Spectrum (UL1TR003142); the National Science Foundation
Graduate Research Fellowship; and the Pat Tillman Fellowship.
D.K. was supported by the Japan Society for the Promotion of Science
(JP21J01690) and the Osamu Hayaishi Memorial Scholarship for Study
Abroad. A.C.F. is supported by the Stanford Graduate Fellowship,
the National Science Foundation Graduate Research Fellowship
Program and the Stanford Lieberman Fellowship. Toshiya Nishimura
was supported by the Japan Society for the Promotion of Science
(JP18K14602 and JP18J00499). A.C.W. was supported by the NIH
(K99HL150218), the Leukemia & Lymphoma Society (3385-19) and the
Edward P. Evans Foundation. J.B. was supported by the International
Postdoc Grant from the Swedish Research Council (2017-00344) and
by the Assar Gabrielsson Foundation.
Author contributions
F.P.S. and D.K. conceived the research, performed most experiments
and analyzed data. Y.N. performed flow cytometry and HSPC
transplant. M.H., J.Z. and I.H. performed ddPCR. K.P. performed the
Nature Biotechnology
https://doi.org/10.1038/s41587-024-02171-w
Southern blot. A.C.F. and Toshinobu Nishimura cloned plasmids. C.T.C.,
J.B., Toshiya Nishimura and A.C.W. analyzed data. H.N., R.M., M.A.K.
and F.P.S. acquired funding and supervised the experiments. H.N. and
R.M. contributed equally. F.P.S. wrote the manuscript, and all authors
edited and approved the final manuscript.
Competing interests
R.M. is on the advisory boards of Kodikaz Therapeutic Solutions,
Orbital Therapeutics, Pheast Therapeutics and 858 Therapeutics.
R.M. is a co-founder of and equity holder in Pheast Therapeutics,
MyeloGene and Orbital Therapeutics. H.N. is a co-founder of and
shareholder in Megakaryon, Century Therapeutics and Celaid
Therapeutics. The remaining authors declare no competing interests.
Additional information
Extended data is available for this paper at
https://doi.org/10.1038/s41587-024-02171-w.
Supplementary information The online version contains supplementary
material available at https://doi.org/10.1038/s41587-024-02171-w.
Correspondence and requests for materials should be addressed to
Fabian P. Suchy, Ravindra Majeti or Hiromitsu Nakauchi.
Peer review information Nature Biotechnology thanks Casey Maguire
and the other, anonymous, reviewer(s) for their contribution to the
peer review of this work.
Reprints and permissions information is available at
www.nature.com/reprints.
Article https://doi.org/10.1038/s41587-024-02171-w

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

Nature Biotechnology
Article
Extended Data Fig. 1 | Comparison of PCR, ddPCR, and Southern blot for
genotyping subcloned colonies after AAV6/Cas9-mediated knockin.
a, Schematic showing location of primers in 3-primer in-out PCR. Primers
span left-homology arm. b, Schematic showing location of primers in 3-primer
in-out PCR. Primers span right-homology arm. c, Schematic showing location
of primers in 2-primer PCR genotyping. Primers span entire editing site.
d, Theoretical gel electrophoresis after genotyping as shown in panel a-c.
WT = wildtype; Mono = monoallelic knockin; Bi = biallelic knockin. e, Gel
electrophoresis from PCR genotyping. 36 subclones expanded after knocking
in CRE into CD14 locus in PT-iPSCs. Ladder size shown in kb. Expected band
sizes shown in panel a-c. Sample number indicated at top and bottom of gels:
L = ladder; N = no-template-control. Interpreted genotype indicated by circle
at top of each gel: white = WT; gray = monoallelic; black = biallelic; red = cannot
be determined. Top, middle, and bottom row of gel indicate PCR-L, PCR-R, and
PCR-F genotyping strategies, respectively. f, Schematic showing ddPCR allele
counting strategy for genotyping. Four ddPCR targets are multiplexed in a single
well for each sample. Primers indicated by arrows, and probes indicated by small
rectangles flanked by primers. Ref-1 and Ref-2 indicate trans references used
to determine overall cell number. RE indicates restriction enzymes. HEX and
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FAM indicate probe color. High and Low indicate concentration of probe (used
for ddPCR amplitude multiplexing), resulting in high or low clusters shown in
panel g. g, Representative two-dimensional ddPCR plot of reaction shown in
panel f. h, References from ddPCR genotyping the 36 samples shown in panel
e. Average of the two references set to 2 copies/cell. Sample # shown at bottom.
i, ddPCR genotyping results. Normalized to references shown in panel h. The
reaction was run +/− restriction enzyme (RE) as shown in panel f. Interpreted
genotype indicated by circle at bottom graph: white = WT; gray = monoallelic;
black = biallelic; red = additional insertions. j, Schematic indicating concatemeric
knockin. Without restriction enzyme, concatemeric inserts will be linked.
k, Schematic indicating ddPCR droplet partitioning. Without restriction enzyme,
linked concatemeric inserts will partition in the same droplet and be counted
only once. l, Schematic showing Southern blot genotyping strategy. RE1 and
RE2 indicate two different restriction enzymes. Probe designed to bind right
homology arm. m, Southern blot of 11 select subclones. Size references
indicated on left in kb. Expected sizes shown in panel l. * indicates off-target
insertion. Interpreted genotype indicated by circle at top of blot: white = WT;
gray = monoallelic; black = biallelic; red = additional insertions.
Article
Extended Data Fig. 2 | Comparison of PCR and ddPCR genotyping at AAVS1
locus. a, Gel electrophoresis from PCR genotyping. 36 subclones expanded
after knocking in Ubc-CRE into AAVS1 locus in PT-iPSCs. Ladder size shown in
kb. Sample number indicated at top and bottom of gels; L = ladder. Interpreted
genotype indicated by circle at top of each gel: white = WT; gray = monoallelic;
black = biallelic; red = cannot be determined. Top, middle, and bottom row
of gel indicate PCR-L, PCR-R, and PCR-F genotyping strategies, respectively.
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b, References from ddPCR. Average of the two references set to 2 copies/
cell. Sample ID shown at bottom. c, ddPCR genotyping results. Normalized
to references shown in panel b. The reaction was run +/− restriction enzyme
(RE). Interpreted genotype indicated by circle at bottom graph: white = WT;
gray = monoallelic; black = biallelic; red = additional insertions or cannot
be determined.
Article
Extended Data Fig. 3 | Characterization of concatemers. a, Concatemer
junction-spanning PCR of 11 select samples (CRE knockin into CD14 locus in
PT-iPSC—same samples as used in Southern blot in Extended Data Fig. 1). Top:
schematic showing location of primers. Size of expected amplicon indicated
assumes viral ITRs are joined end-to-end. Bottom: gel electrophoresis of PCR
products. Top of gel indicates genotype previously determined by ddPCR
and Southern blot. Bottom of gel indicates sample number. L = ladder;
N = no-template control. Ladder size indicated in kb. F and R indicate forward and
reverse primers, respectively. b, Repeat of panel a with forward primer only.
c, Repeat of panel a with reverse primer only. d, Schematic showing sequence of
chimeric ITR mapped to regions of viral vector genome. Green triangle indicates
AhdI restriction enzyme cutsite. Orange arrows and rectangle indicate primer
and probe sites for ITR ddPCR or qPCR (used in panel g-k). e, References from
ddPCR analysis of 36 subclones (same samples as in Extended Data Fig. 1).
Average of the two references set to 2 copies/cell. Sample number shown
at bottom. References are in cis: Ref-1 is upstream (5′) of LHA and Ref-2 is
downstream (3′) of RHA. f, ddPCR results of counting LHA and RHA. Analyzed
+/− restriction enzyme (RE) used to separate concatemeric regions. Normalized
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to references shown in panel e. g, qPCR measurement of AAV ITR when analyzed
+/− AhdI RE. Y-axis is fluorescent intensity; x-axis is cycle number. Left graph
shows DNA extracted from sample #10 (concatemeric monoallelic KI). Right
graph shows DNA extracted from sample #14 (monoallelic KI by end-joining).
Δ indicates change in cycle threshold when run with and without AhdI. h, qPCR
measurement of control primer-probe set Cis Ref-1. Same samples and axis as
panel g. i, ddPCR results of counting ITR insertions in the 36 subclones +/− RE
(+RE is AhdI and MseI). Normalized to cis-references (not shown). j, Schematic
showing ddPCR linkage analysis of ITR and cis-references. Left indicates
concatemeric knockin with chimeric ITR inserted. Right indicates end-joining-
mediated knockin. Legend at bottom. Dashed lines indicate amplicons are linked.
Red X indicates linkage is lost when adding RE. k. Linkage heat map between
cis-reference sites and ITRs measured by ddPCR (analyzed with addition of AhdI).
Sample number shown at top. Amplicon sites and legend shown in panel j. Top
row indicates the % of Ref-1 sites bound to an ITR; middle row indicates linkage
between Ref-2 sites bound to an ITR; bottom row indicates the % of Ref-1 sites
bound to Ref-2 sites.
Article
Extended Data Fig. 4 | Characterization of dual-knockin subclones.
a, Schematic indicating strategy for genotyping double knockin subclones
using ddPCR linkage analysis. Cis reference sites shown as small light-gray and
dark-gray squares. Ubc-GFP amplicon site indicated by small green square.
Ubc-mCherry amplicon site indicated by small red square. Dashed-lines
indicate linkage, which can be measured by ddPCR. b, Schematic indicating
strategy for counting concatemers in double knockin subclones using ddPCR.
Scissors indicate restriction enzymes. Red X indicates linkage lost, such
that concatemeric inserts will be separated. c, Images of 72 subclones after
dual knocking of Ubc-GFP and Ubc-mCherry into the TET2 locus in PT-iPSCs.
Subclones were selected and expanded as shown in Fig. 2e. Left and right side
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show the same images, processed differently. The left images were processed
using auto-contrast prior to stitching. The right images were processed with
uniform settings. The upper 24 colonies were selected from the low-gate during
FACS and the lower 48 colonies were selected from the high gate as shown in
Fig. 2f. White scale bar (lower right) indicates 400 microns. d, Correlation of GFP
mean fluorescent intensity (MFI) and copies of Ubc-GFP per cell. MFI measured
from uniformly processed images. Ubc-GFP copies/cell measured as shown
in panel b. Dashed line indicates linear regression—equation and R2 indicated
on graph. e, Correlation of mCherry MFI and copies of Ubc-mCherry per cells
(similar to panel d).
Article
Extended Data Fig. 5 | Flow cytometry plots and editing rate in human PSCs
+/−ITR removal. a-g, Flow cytometry plots 5 days after double knockin of
Ubc-GFP and Ubc-mCherry into human PSCs. Loci (TET2, AAVS1, HBB, RUNX1)
and cell line (PT-iPSC, WT-iPSC, H9) shown at top. NC indicates no cut (ITR not
removed). IC and DC indicate internal cut and distal cut methods for ITR removal,
respectively. GFP and mCherry fluorescent intensity indicated on x- and y-axis,
respectively. Gating indicated by quadrants; % cells in each quadrant shown on
plot. Double positive cells were sorted into individual wells and used for later
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clonal analyses. h, Change in polydispersity in flow cytometry quadrants in
panel a-g (n = 7, each panel in a-g is one measurement). Q1-Q4 indicate upper left,
upper right, lower right, and lower left quadrant, respectively. Polydispersity
measured as absolute average deviation and shown relative to NC. Error bars
indicate 1 standard deviation; center indicates mean. * indicates P = 0.02 to 0.05.
** indicates P = 0.005. P values are from two-sided paired t-test. i, Legend for
panel j-k. j-k, % cells positive for GFP and/or mCherry ( j), and % double positive
(GFP and mCherry, k), as indicated by panel a-g.
Article
Extended Data Fig. 6 | Concatemer rate at various MOIs. a, Schematic
indicating ddPCR linkage analysis strategy for measuring KI frequency in bulk
(non-subcloned) samples. Cis reference sites shown as small light-gray and
dark-gray squares. Ubc-GFP amplicon site indicated by small green square.
Dashed-lines indicate linkage, which can be measured by ddPCR. ID1 and ID2
are small (<100 bp) unique DNA sequences added outside the homology arms
in the viral vector genome as shown at the top. b, Schematic indicating ddPCR
linkage analysis strategy for measuring concatemeric KI frequency in bulk
(non-subcloned) samples. Similar depiction as panel a. ddPCR amplicons for
ID1 and ID2 shown as dark purple squares. c, Flow cytometry plots 5 days after
KI of Ubc-GFP into the HBB locus in H9 ESCs. Editing performed at various
MOIs indicated by number on each plot. Right gate on each plot indicates the
positive population, which was sorted and expanded for ddPCR analysis. Side-
scatter-area on y-axis and GFP fluorescent intensity on x-axis. Gating indicated
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by trapezoid; % cells in each gate shown on plot. d, Flow-chart indicating
subpopulations analyzed for panel e-h and j-m. e, Editing rate (%GFP positive
cells) at various MOIs as indicated by flow cytometry in panel c. f, Knockin rate
(%HBB alleles with GFP knockin) at various MOIs as indicated by ddPCR. Black line
indicates measurements performed by ddPCR linkage analysis as shown in panel
a. Blue dashed-line indicates measurements performed by ddPCR allele counting
as shown in Extended Data Fig. 1f. Samples analyzed were GFP+ cells sorted and
expanded at each MOI as shown in panel c. The same samples are used for ddPCR
analysis in panel g-h. g, Concatemeric knockin rate (%GFP knockin alleles with
concatemer) as indicated by ddPCR. h, Average size of concatemeric knockin
(for example, 1 = single KI, 2 = a concatemeric knockin with a single repeat, etc.)
as indicated by ddPCR. i, Flow cytometry plots 5 days after KI of Ubc-GFP into the
IL2RG locus (x chromosome) in H1 (male) ESCs. Similar to panel c. j-m, Similar to
panel e-h. Samples analyzed are from panel i.
Article
Extended Data Fig. 7 | Comparison of HDR- and NHEJ-mediated knockin rate
+/−ITR removal. a-d, In-out PCR and gel electrophoresis of subclones after dual-
knockin of Ubc-GFP and Ubc-mCherry into the TET2 locus in PT-iPSCs. Subclones
were selected from NC, IC, and DC groups that contained biallelic knockins
without concatemers (determined by ddPCR). Primer location and expected band
size shown on schematic at top of each gel. Larger than expected bands, indicated
by *, could result from NHEJ-mediated knockin. e, Schematic showing strategy for
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comparing HDR- and NHEJ-mediated knockin rate by flow cytometry. Mismatched
homology arms (HA) measure NHEJ-mediated knockin. Matching homology arms
measure HDR-mediated knockin rate. f, Results of editing with matched versus
mismatched homology arms as measured by flow cytometry. Number at top of
graph corresponds to schematic in panel e. Experiment was performed with the
IC method of ITR removal (orange bars) or no removal (gray bars). GFP+ cells
measured by flow cytometer more than 2 weeks after editing.
Article
Extended Data Fig. 8 | Flow cytometry plots and editing rate in human HSPCs
+/−ITR removal. a-d, Flow cytometry plots 5 days after double knockin of Ubc-
GFP and Ubc-mCherry into human CD34+ HSPCs. Loci (TET2, HBB) and repeat
number shown at top. NC indicates no cut (ITR not removed). DC indicates distal
cut method for ITR removal. GFP and mCherry fluorescent intensity indicated
on x- and y-axis, respectively. Gating indicated by quadrants; % cells in each
quadrant shown on plot. Double positive cells were sorted into individual wells
and used for later clonal analyses. e-g, Flow cytometry plots 5 days after knockin
of Ubc-GFP into in male human CD34+ HSPCs at the IL2RG locus (x chromosome).
GFP fluorescent intensity indicated on x-axis; side-scatter-area on y-axis.
Gating indicated by rectangles; % cells in each gate shown on plot. GFP positive
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cells were sorted into single wells and used for later clonal analyses. h-i, Flow
cytometry plots 5 days after knockin of Ubc-GFP in human CD34+ HPSCs at the
HBB locus. Gating indicated by rectangles; % cells in each gate shown on plot. GFP
positive cells were sorted and transplanted into mice for later analyses. j, Table
summarizing HSPC analyses. Legend for panel k-m. k, % cells positive for GFP
and/or mCherry as indicated by panel a-i. l, % cells positive for GFP and mCherry
as indicated by panel a-d. m, % CD45+ GFP+ human cells in mouse whole bone
marrow 4 months after transplantation. Transplanted cells initially sorted from
panel h-i and transplanted into 2 mice each (n = 8 total mice). Error bars indicate
1 standard deviation; center indicates mean.
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μ
μ
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