Research

DNA methylation can alter CRISPR/Cas9 editing frequency and

DNA repair outcome in a target-specific manner
Adela Pribylova1,2 , Lukas Fischer2 , Douglas E. Pyott3 , Andrew Bassett4 and Attila Molnar1
1
Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh, EH9 3BF, UK; 2Faculty of Science, Charles University, Prague 128 44, Czech Republic; 3The Wellcome Trust
Center for Cell Biology, Institute of Cell Biology, The University of Edinburgh, Edinburgh, EH9 3BF, UK; 4Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, CB10 1SA,
UK

Summary
Author for correspondence:
The impact of epigenetic modifications on the efficacy of CRISPR/Cas9-mediated double-
Attila Molnar stranded DNA breaks and subsequent DNA repair is poorly understood, especially in plants.
Email: attila.molnar@ed.ac.uk
In this study, we investigated the effect of the level of cytosine methylation on the outcome
of CRISPR/Cas9-induced mutations at multiple Cas9 target sites in Nicotiana benthamiana
Received: 11 March 2022 leaf cells using next-generation sequencing.
Accepted: 2 May 2022

We found that high levels of promoter methylation, but not gene-body methylation,
decreased the frequency of Cas9-mediated mutations. DNA methylation also influenced the
New Phytologist (2022) 235: 2285–2299 ratio of insertions and deletions and potentially the type of Cas9 cleavage in a target-specific
doi: 10.1111/nph.18212 manner. In addition, we detected an over-representation of deletion events governed by a
single 50 -terminal nucleotide at Cas9-induced DNA breaks.

Our findings suggest that DNA methylation can indirectly impair Cas9 activity and subse-
Key words: Cas9, chromatin, DNA
methylation, DNA repair, microhomology- quent DNA repair, probably through changes in the local chromatin structure. In addition to
mediated, next-generation sequencing, the well described Cas9-induced blunt-end double-stranded DNA breaks, we provide evi-
nonhomologous end joining, staggered end. dence for Cas9-mediated staggered DNA cuts in plant cells. Both types of cut may direct
microhomology-mediated DNA repair by a novel, as yet undescribed, mechanism.

to as microhomologies present on both 30 DNA ends close to the

Introduction
CRISPR/Cas9 has been extensively used in gene editing in eukary-
otes, and many of the mechanistic details of Cas9-induced double-
stranded breaks (DSBs) have been well described. Cas9-mediated
cleavage mostly occurs as a blunt DSB three nucleotide upstream
of the protospacer adjacent motif (PAM) (Jinek et al., 2012;
Josephs et al., 2015). However, animal and yeast studies indicate
that the Cas9 can also induce staggered DSBs with single 50
nucleotide overhangs (Li et al., 2015; Lemos et al., 2018).
Double-stranded breaks can be repaired by several endogenous
DNA repair mechanisms. In eukaryotes, the predominant mecha-
nism in differentiated cells is classical nonhomologous end joining
(c-NHEJ), in which blunt DNA ends are directly re-ligated. Alter-
natively, short overhangs, resulting for example from CRISPR/
Cas9 staggered cuts, can be filled in and then ligated forming short
insertions (IN) (Beying et al., 2021). Double-stranded breaks can
also be repaired through homology-directed repair. When the
repair relies on extensive nucleotide sequence similarity/homology
(such as 20–100 bp) at 30 DNA ends it comprises two main path-
ways: single-strand annealing and homologous recombination
(Roth et al., 2012; Bothmer et al., 2017; Beying et al., 2021). The
remaining dominant pathway is microhomology-mediated end
joining (MMEJ), also known as alternative end joining (Alt-EJ),
which relies on annealing of short complementary regions referred
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distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
break, which can result in short deletions (DEL) (Chiruvella et al.,
2013; Beying et al., 2021; Vu et al., 2021).
Successful CRISPR/Cas9-induced mutagenesis is conditioned
by the selection of an appropriate target sequence. Several bioin-
formatics tools have been developed to aid the selection of opti-
mal sequences to be targeted by Cas9. Many of these algorithms
provide predicted mutagenic efficiencies (on-target scores) and, if
a whole-genome sequence is available, some programs will also
provide predictions of off-target sequences (Liu et al., 2017; Cui
et al., 2018). While these algorithms facilitate the design of
gRNAs, the predicted on-target efficiencies can often be inaccu-
rate (Naim et al., 2020), indicating that our understanding of the
principles underlying Cas9 binding and cleavage of target DNA
and subsequent repair of DSB in vivo is far from complete. A
possible reason for the discrepancies between the in silico predic-
tions and experimental results could be that most of the gRNA
design tools do not consider the epigenetic status of the target
locus. It is likely that the chromatin structure of a locus (in addi-
tion to the underlying base sequence) will be an important deter-
minant for the efficacy of Cas9 binding, cleavage and subsequent
repair. Indeed, there have been reports that the frequency of
Cas9-induced mutagenesis is lower for heterochromatin com-
pared with euchromatin (Chen et al., 2016; Daer et al., 2017,
2018). However, other reports have suggested that Cas9-induced
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mutation frequencies are comparable for heterochromatin and
euchromatin (Yu et al., 2013; Feng et al., 2016; Kallimasioti-Pazi
et al., 2018). This indicates that epigenetic factors influencing the
CRISPR/Cas outcomes are more complex than just considering
the broad heterochromatin/euchromatin dichotomy (Schep et al.,
2021). For instance, it has been shown that the epigenetic avail-
ability of the PAM sequence is crucial to the action of Cas9, such
that PAMs associated with nucleosomes cannot be located by
Cas9 (Hinz et al., 2015; Horlbeck et al., 2016; Yarrington et al.,
2018). However another epigenetic effect that could influence
the ability of Cas9 to find and cleave DNA is the methylation sta-
tus of the target locus. While DNA methylation at the PAM was
found to have no effect on Cas9 activity in human cell lines (Hsu
et al., 2013; Fujita et al., 2016), this phenomenon is yet to be
tested in other organisms.
Here we present a detailed study on Nicotiana benthamiana in
which we use Tobacco Rattle Virus (TRV) as a molecular switch
to change the chromatin state of a reporter gene from an actively
transcribed to a transcriptionally silenced state. Unlike previously
used systems, our unique approach enabled us to interrogate dif-
ferent chromatin states of the same locus with the same set of
CRISPR/Cas9 genome editing reagents and systematically
describe the effect of chromatin state on the frequency and type
of mutations induced at various Cas9 targets in a huge set of
independently edited cells.
Materials and Methods
pt, pb, mt, mb, dt, db, utg, mbg, dtg; Table S2) were verified by
Sanger sequencing. The BASTA resistance cassette (CaMV35S::
bar::nos) was deleted from the original destination binary vector
pDe_CAS9 (Fauser et al., 2014) by HindIII restriction enzyme
digestion and re-ligation to avoid interference with the 35S
sequence. The gRNA expression cassettes were then recombined
into the modified destination vector (pDe_CAS9_no_basta) by
Gateway cloning (LR reaction) according to the manufacturer’s
instruction (Life Technologies, Carlsbad, CA, USA), which
yielded the pDE_CAS9_no_basta_x binary vectors (Table S2) for
plant transformation.
Plant material
Wild-type N. benthamiana plants, N. benthamiana 16c line
expressing the green fluorescent protein (GFP) reporter gene
under the constitutive CaMV 35S promoter (Ruiz et al., 1998)
and its transcriptionally silenced progeny (S1: the virus-free
progeny of TRV-35S-infected 16c; Fig. 1) (Fei et al., 2021) were
grown in SANYO/Panasonic growth chambers with 16 h : 8 h,
light : dark photoperiods at 21°C under 150 lmol m2 s1 light.
Viruses and virus sap
Wild-type TRV, recombinant TRV-35S and TRV-GFP, and
virus sap collection from N. benthamiana plants have been
described in Fei et al. (2021).

Generation of CRISPR/Cas9 gene-editing constructs Transient expression of gene-editing reagents in planta

Cas9 target sites were identified by GENEIOUS v.11.1.5 (http://
www.geneious.com) (Kearse et al., 2012) and potential off-
targets were analysed by Sol Genomics Network BLAST (https://
www.solgenomics.net/tools/blast) (Fernandez-Pozo et al., 2015)
and CRISPR RGEN CAS-OFFINDER (http://www.rgenome.net/
cas-offinder) (Bae et al., 2014). The corresponding oligonu-
cleotides with BbsI compatible overhangs (Supporting Informa-
tion Table S1) were annealed into a dsDNA duplex,
phosphorylated with T4 Polynucleotide Kinase (New England
BioLabs, Hitchin, UK) and subsequently cloned into BbsI-di-
gested entry vector pEn_Chimera (Fauser et al., 2014). The
resulting gRNA expression cassettes (pEn_Chimera_x; x = ut, ub,
Agrobacterium tumefaciens AGL1 strain was transformed with
pDe_Cas9_no_basta_x vectors (Table S2) by electroporation and
selected on solidified LB plates using 100 µg ml1 spectinomycin
and 50 µg ml1 rifampicin. For each construct, 200 µl of overnight
starter culture was added to 10 ml LB supplemented with the above
antibiotics and incubated for 24 h at 28°C and 11 g. The bacterium
culture was then centrifuged at 3000 g for 10 min, and the pellet
was resuspended in infiltration medium (IM, 10 mM MES (pH
5.6), 300 µM acetosyringone, 10 mM MgCl2) and incubated for
3–4 h at room temperature. The optical density was adjusted to 1.5
and 0.15 OD600 by adding a fresh IM buffer. An Agrobacterium
suspension was infiltrated with a 1-ml syringe into the abaxial side

Fig. 1 Virus-induced epigenetic switch system. (a) Schematic diagram of the GFP reporter gene under the control of the constitutive CaMV promoter P35S.
(b) Schematic diagram of Tobacco rattle viruses (TRV): wild-type virus (TRV-WT) and its derivatives harbouring a 120-bp segment from P35S (TRV-35S)
and GFP (TRV-GFP). (c) Scheme of the four plant conditions used for the analysis of epigenetic changes at the 35S promoter. Noninfected and TRV-WT-
infected plants contain transcriptionally active euchromatin, while the TRV-35S-infected plants and their noninfected progeny (S1) have induced chromatin
silencing at the 35S promoter. (d) Scheme of GFP expression for the different plant conditions. 16c plants express GFP; TRV-WT plants have partially, non-
specifically reduced GFP expression due to ongoing virus infection; TRV-35S-infected plants exhibit reduced GFP expression due to TRV-35S-mediated for-
mation of sRNAs that trigger dense cytosine methylation in the target 120-bp region of P35S; S1 plants have a slightly reduced GFP expression due to
inherited DNA methylation at the 35S promoter. (e) Fluorescence of GFP in representative plants of the different plant conditions, imaged under UV light.
Pictures of plants from all biological replicates can be found in Supporting Information Fig. S1. (f) Quantification of GFP mRNA and TRV RNA by RT-qPCR
for the different plant conditions. **, * and ns represent significant difference for P < 0.01, P < 0.05 and not significant, respectively. Individual pairs were
analysed by two-tailed t-test with a = 0.05. Error bars represent standard deviations. (g) Average of methylated cytosines in the 120-bp target region in
individual epigenotypes and sequence contexts from three independent biological replicates. (h) DNA cytosine methylation of P35S in detail for the four
plant conditions. A top schematic diagram of P35S shows the position of the TRV-35S target and positions of CRISPR/Cas9 target sites (colour-coded
CRISPR/Cas9 target sites ut, ub, pt, pb, mt, mb, dt, db are explained in Fig. 2). Only cytosines that are present in the leading strand are shown. nd, not
done; the bottom bar is the sequence context of individual cytosines.

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of three leaves (4th–6th youngest leaves) of 5-wk-old N. benthami- and the plants were kept under standard conditions (described in
ana. For agro-infiltrating virus-infected plants, the first and the sec- ‘Plant material’ in the Materials and Methods section) for another
ond leaf of 3-wk-old N. benthamiana were rub-inoculated with the 2 wk before agro-infiltration. All experiments were independently
corresponding virus sap as previously described (Fei et al., 2021) repeated three to five times.

(a) (b)
P35S GFP NosT TRV- WT TRV- 35S TRV- GFP

Stably
(c) Euchromatin silenced chromatin (d)
* * * *
* ** ** * * **
Virus absent

mRNA mRNA

P35S GFP T
**
P35S target GFP T
**
Non-infected S1 Non-infected S1
+T
RV
-35 Next
+TRV-WT S generation

TRV-WT TRV-35S
Virus present

TRV- WT TRV- 35S

TGS iral
siRNAs
mRNA
TGS *
x
Induced P35S GFP T
* ** *
P35S target GFP T
Euchromatin silenced chromatin
****
* *** *******
** ** TRV-WT TRV-35S
***** **************
*

(e) (f) (g)

Non-infected

* 40 80%

methylated cytosines
15
S1

30 Average of 60%
GFP/EF1α

TRV/EF1α

10 ** 20 40%

5 10 20%
RV-35S
RV-WT

0 0 0%
TRV

TRV

TRV
RV-WT TRV
RV-35S non-infected S1 *CHH *CHG *CG

(h) 120 bp target 120 bp target

P35S P35S
Non-infected

1 1 nd
S1

2 2 nd

3 3 nd
TRV-35S
TRV-WT

1 1
2 2
3 3

No methylation *CHH *CG *CHG

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Imaging GFP fluorescence
GFP expression was regularly monitored under UV light using a
handheld mercury UV lamp (UVP Blak-RayTM B-100AP High-
Intensity UV Inspection Lamp, Upland, CA, USA). Photographs
were taken using a Canon G16 camera as described previously
(Fei et al., 2021).
Sample collection
A leaf disc (8 mm in diameter) was cut out from each infiltrated leaf
(4th, 5th and 6th) and combined into a single biological sample 5 d
post infiltration (dpi). Three samples were collected from each plant,
frozen in liquid nitrogen and stored at 80°C until further use.
Mutation analysis
DNA was isolated using the DNeasy Plant Mini Kit (Qiagen). A
T7 endonuclease assay was carried out as previously described
(Pyott et al., 2016). For assessing the frequency of gene editing,
the Cas9 targeted loci were amplified with the corresponding
primers (Table S1), and the resulting DNA was cleaned up using
SAP-ExoI digestion (NEB). Sanger sequencing was performed
using the BigDye Terminator v.3.1 Cycle Sequencing Kit
(ThermoFisher Scientific, Waltham, MA, USA) and completed on
an ABI 3730XL instrument by Edinburgh Genomics (Edinburgh,
UK). The efficacy of gene editing was calculated using TIDE
(https://tide.nki.nl/) (Brinkman et al., 2014). For creating a next-
generation sequencing (NGS) library, the target loci were ampli-
fied with the corresponding Illumina compatible primers
(Table S1), which were subsequently barcoded using the Nextera
XT Index Kit v.2 (Illumina, San Diego, CA, USA). The NGS
library was sequenced on an Illumina MiSeq instrument. The data
were analysed using the CRISPRESSO2 tool (Clement et al., 2019)
followed by additional analysis using in-house PYTHON scripts (re-
lease 3.7.5) and MS EXCEL 365 (Microsoft, Redmond, WA, USA).
RNA analysis
RNA was isolated using the RNeasy Plant Mini Kit (Qiagen).
After Turbo DNase treatment (Ambion), cDNAs were made
from 0.5 lg RNA using random hexamers primers and Super-
script III (Life Technologies). Quantitative RT-PCR was per-
formed using the SYBR Green I Master Mix on a
LightCycler480 (Roche) using gene-specific primers (Table S1).
Data were converted with LC480 software (v.2014.1), and Ct val-
ues were calculated using LINREGPCR (v.2012.0) (Ramakers
et al., 2003), both software are available at https://www.
medischebiologie.nl/files/. The analysis was carried out in MS
EXCEL 365 (Microsoft). Three technical replicates were per-
formed for each biological replicate.
DNA methylation analysis
Genomic DNA was converted using the EZ DNA Methylation-
Gold Kit (Zymo Research, Irvine, CA, USA). Target loci were
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amplified using bisulfite PCR primers (Table S1) and EpiMark®
Hot Start Taq DNA Polymerase (NEB). The resulting PCR
products were ligated into the pGEM®-T Vector (Promega),
which were then used to transform Escherichia coli (DH5a) cells.
In total, 8–10 clones from each sample were sequenced using
BigDye 3.1, as described previously. DNA methylation patterns
were analysed using GENEIOUS v.11.1.5 (http://www.geneious.
com) (Kearse et al., 2012).
Statistical analysis
Statistical analyses were performed using the NCSS 9 and SPSS 28
statistical software. Significance level a was always set to 0.05. All
variables were checked for outliers, and assessed for normality
using the Shapiro–Wilk test and Levene’s test for equal variance.
A one-tailed or two-tailed two-sample independent t-test was
used in which the two independent groups were compared, based
on expectations from the groups being compared. To determine
whether the independent variables had an interaction, two-way
analysis of variance (ANOVA) tests were performed, followed by
Tukey’s post hoc test if possible.
Results
Virus-induced gene silencing can be used as an ‘epigenetic
switch’ to alter the cytosine methylation status of a target
locus
To test how chromatin state affects the efficacy of CRISPR/Cas9
mutagenesis in planta, we developed a system that allowed us to
change the chromatin state of a single locus and subsequently
analyse CRISPR/Cas9-induced mutations at that locus. In this
system, we made use of a recombinant RNA virus to induce
DNA methylation by triggering the plant’s RNA-dependent
DNA methylation (RdDM) pathway at the target locus. The
RdDM pathway can be triggered by some viruses when short
interfering RNAs (siRNAs) are produced by the plant’s antiviral
RNA silencing response. Certain classes of these virus-derived
siRNAs can direct plant-encoded DNA methyl transferases to
methylate cytosine residues of DNA that bear sequence comple-
mentarity to the siRNA. Plant viruses can be turned into virus-
induced gene silencing (VIGS) vectors if sequences present in the
plant’s genome are cloned into the viral genome. Infection with
such viruses will result in the production of siRNAs that are com-
plementary to the region of the plant’s genome that was inserted
into the virus. If a promoter sequence is inserted into the VIGS
vector, the siRNA-mediated RdDM can lead to cytosine methy-
lation of that promoter and consequently result in transcriptional
gene silencing (TGS) (Jones et al., 2001; Pribylova et al., 2019).
For our experimental system, we utilised a previously described
and well characterised transgenic N. benthamiana line (named
16c) that contained a single-copy genomic insertion of the GFP
reporter gene under the constitutive 35S promoter (Fig. 1a) (Ruiz
et al., 1998). In the 16c line, the 35S::GFP cassette is highly
expressed, implying that the transgenic cassette is integrated into
an epigenetically active region of the plant’s genome. In a
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previous study (Fei et al., 2021), we adapted a TRV VIGS vector
(Jones et al., 2001) to induce RdDM of the 35S promoter by tar-
geting a short segment (120 bp) upstream of the transcription
start site and therefore transcriptionally silence the expression of
GFP in 16c plants. We named this vector TRV-35S (Fig. 1b). To
control for possible effects of the virus infection itself (unrelated
to the VIGS-induced RdDM of the target) we used a TRV vector
without the 120-bp section of the 35S promoter inserted and
named this vector TRV-WT (Fig. 1b). Furthermore, we collected
seeds from the TRV-35S-infected plants to allow us to evaluate
the effects of cytosine methylation at the target locus in plants
containing no viral RNA. The basis of this is that TRV transmis-
sion to progeny seedlings was extremely low (< 1%) (Jones et al.,
2001) but cytosine methylation that is in a symmetrical sequence
context (i.e. CG or CHG, where H represents A, C or T) can be
maintained through both mitotic and meiotic cell divisions and
exhibits transgenerational inheritance (Jones et al., 2001). We use
the term S1 to refer to these plants grown from the seeds col-
lected from TRV-35S-infected plants as they are the first-
generation progeny of the transcriptionally silenced plants
(Fig. 1c). Therefore, our system allows for the comparison of two
different epigenotypes (high/low cytosine methylation) at the
35S promoter in the context of virus-infected and nonvirus-
infected tissue (Fig. 1d).
First, we tested the efficacy of our TRV-based ‘epigenetic
switch’. When imaged under UV light, 16c plants that had been
rub-inoculated with TRV-35S glowed red due to the autofluores-
cence of chlorophyll and the green glow of GFP was completely
absent. By contrast, for both the noninfected and TRV-WT-
infected 16c plants a green GFP signal was discernible and was
particularly visible in the upper leaves and petioles. A weak GFP
signal could be seen in the S1 plants, indicative of only a partial
silencing of GFP (Figs 1e, S1). This silencing of the GFP expres-
sion was confirmed in three randomly selected independent bio-
logical replicates by RT-qPCR. TRV-WT-infected plants had a
significantly higher level of GFP mRNA compared with TRV-
35S-infected plants (P = 0.006, two-tailed t-test), which had neg-
ligible levels of GFP mRNA. The noninfected 16c plants had
more than twice the level of GFP mRNA than S1 plants
(P = 0.011, two-tailed t-test), but there was still a weak expression
of GFP mRNA in the S1 plants (Fig. 1f). Moreover, we did not
observe any difference in the abundance of TRV RNA between
the TRV-35S and TRV-WT infections (P = 0.441, two-tailed t-
test).
To confirm that the basis of the observed GFP silencing was a
result of VIGS-induced RdDM, we performed bisulphite
sequencing. DNA samples taken from TRV-WT-infected plants
showed almost no methylation in the 120-bp target region, and
weak methylation was observed in the upstream promoter region,
mainly in the CG context. By contrast, DNA samples from
TRV-35S-infected plants showed strong methylation in the tar-
get region with a weak spreading of methylation towards 30 and
50 ends of the 35S promoter, independent of the C sequence con-
text. In the upstream part of the promoter, we observed increased
methylation in the symmetrical CG and CHG context, mainly in
the region where even TRV-WT-infected plants showed some
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Research 2289
CG methylation. As expected, noninfected plants showed a simi-
lar methylation pattern as TRV-WT-infected plants: no methyla-
tion in the target region and limited methylation in the upstream
region of the promoter. S1 plants inherited an increased level of
mostly symmetric CG and CHG methylation within the target
region of the 35S promoter, but only at some C positions
(Fig. 1g,h). Taken together, our data show that infection with
TRV-35S can switch the 35S::GFP transgene of 16c plants from
an epigenetically active to an epigenetically inactive (silenced)
state due to virus-induced methylation of the 35S promoter and
that this silenced epigenotype is partially inherited to the virus-
free progeny.
The efficacy of CRISPR/Cas9 editing is inversely correlated
with the level of cytosine methylation for targets within a
promoter region
Having demonstrated that we could alter the cytosine methyla-
tion status of a promoter by VIGS, we sought to use this as a tool
to determine how DNA methylation at a given locus might influ-
ence the frequency of CRISPR/Cas9-induced mutations
(Fig. 2a). To this end, we designed CRISPR gRNAs that would
direct Cas9 to the region of the 35S promoter that we had tar-
geted for RdDM using TRV-35S (Fig. 2b). Additionally, we
designed gRNAs to direct Cas9 to a region of the 35S promoter
that was upstream of the TRV-35S targeted region but still
showed an increase in VIGS-induced cytosine methylation
(Figs 1h, 2b). Within the TRV-35S targeted region of the pro-
moter, we designed gRNAs to direct Cas9 to three positions
which we referred to as proximal, middle and distal (based on
their position relative to the start of the promoter). For each of
the four regions of the 35S promoter (namely: upstream, proxi-
mal, middle and distal) we designed pairs of gRNAs that would
anneal to each of the two complementary strands of the target
DNA, which for convenience we referred to as the top and bot-
tom strands. As far as possible we designed these pairs of gRNAs
so that they would overlap, although we could not obtain a 100%
overlap of the gRNAs directed to the top and bottom DNA
strands due to the constraints of PAM availability. For ease of ref-
erence, we named each of the gRNAs using acronyms to describe
both their position in the 35S promoter (upstream/proximal/
middle/distal) and their cognate DNA strand (top/bottom).
Therefore, we named the eight gRNAs ut, ub, pt, pb, mt, mb, dt,
db (Fig. 2b).
To test the in planta activity of these gRNAs, we cloned them
individually into the pDe-Cas9 binary vector under the control
of the Arabidopsis thaliana U6 promoter. This binary vector also
contained a Cas9 expression cassette driven by the constitutive
Ubiquitin4–2 promoter from Petroselinum crispum (PcUbi4-2)
(Fauser et al., 2014). Syringe infiltration of A. tumefaciens cul-
tures containing these gRNA and Cas9 expression vectors was
used to transiently transform the leaves of 16c plants with these
CRISPR/Cas9 constructs. To optimise the conditions for
A. tumefaciens-mediated delivery of these constructs, we per-
formed each infiltration at two different densities of bacterial sus-
pension (namely OD600 = 1.5 and OD600 = 0.15). DNA was
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(a) 2. Introduction of CRISPR/Cas9

reagents by A. tumefaciens
1. Controlling epigenetic
state by TRV infection

3. DNA extraction and analysis of

the Cas9 mutagenesis efficacy

(b) dtg

P35S Target GFP NosT

120bp region of 35S promoter targeted for RdDM by TRV-35S

–500 ut –480 –200 pt –180 –160 mt –140 –120 dt –100 – 80

P35S
ub pb mb db
(c)
60%
ns ns ns
* * ns
* * 60%
ns ns ns ns ns ns ns ns
Mutation frequency

Mutation frequency

50% 50%
(NGS OD 0.15)

(NGS OD 0.15)

40% 40%

30% 30%

20% 20%

10% 10%

0% 0%
ut ub pt pb mt mb dt db ut ub pt pb mt mb dt db
TRV
RV-WT TRV
RV-35S Non-infected S1
Fig. 2 The impact of epigenotype on CRISPR/Cas9-induced mutation frequency. (a) Summary of the experimental design. Here, 3-wk-old 16c plants were
infected with the Tobacco Rattle Virus (TRV) constructs. Noninfected plants were used as controls. At 2 wk later, the newly emerging leaves were infiltrated
with Agrobacterium tumefaciens carrying the corresponding CRISPR/Cas9 constructs. Samples were collected 5 d post infiltration (dpi) and were subse-
quently analysed for the efficacy of CRISPR/Cas9-mediated gene editing. (b) Schematic diagram of the CRISPR/Cas9 target sites at the P35S::GFP::NosT
locus. gRNAs were named according to the position of the protospacer adjacent motif (PAM) sequence (circled). t, top; b, bottom; u, upstream; p, proxi-
mal; m, middle; d, distal. The region of P35S targeted for induced cytosine methylation is indicated with a black line. (c) CRISPR/Cas9-induced mutation
frequencies in plants infected with TRV-WT or TRV-35S (left panel), and in noninfected 16c and TRV-35S-noninfected progeny (S1) plants (right panel).
Individual points (o, x, □) represent next-generation sequencing (NGS) data (OD600 = 0.15) from three independent biological replicates. Lower range,
box, upper range and a horizontal line represents the first quartile, second and third quartile, fourth quartile and median, respective. Two-way ANOVA was
used to examine the effect of the target position and epigenotype on mutation frequency and showed a statistically significant interaction between the tar-
get position and the epigenotypes (F(7, 32) = 3.017, P = 0.015) in infected plants. By contrast, the interaction was not significant for noninfected plants (F
(7, 32) = 0.960, P = 0.477). For both groups, infected and noninfected, the simple and main effect of the target site was significant (P < 0.001). To examine
the effect of the epigenotype in the individual target site, a one-tailed t-test was used, as individual target sites had only two independent variables. * and
ns indicate significant difference for P < 0.05 and not significant, respectively.

extracted from the infiltrated leaves and a T7 assay was performed
to semiquantitatively assess the mutagenic capacity of each of the
gRNA-containing constructs. Encouragingly, the target ampli-
cons digested with T7 endonuclease produced the expected cleav-
age products for all of the gRNA constructs, indicating that all of
the gRNAs are capable of directing Cas9 to the respective target
loci to induce mutations using both the high and the low bac-
terium densities (Fig. S2A).
Next, we used our VIGS system to induce methylation at the
35S promoter to determine any effects of this induced

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methylation on the mutagenesis frequency of each of the gRNAs.
Here, 16c plants were infected with either TRV-35S or TRV-
WT and were incubated for 2 wk to allow the infection to
become systemic and initiate RdDM. Newly formed leaves of
these systemically infected plants were then infiltrated with the
different gRNA-containing CRISPR/Cas9 constructs (Fig. 2a).
In parallel, noninfected plants and S1 plants were infiltrated with
the same CRISPR/Cas9 constructs to analyse the active/silenced
epigenotypes in the absence of TRV infection. DNA was then
extracted from the infiltrated leaves to determine the rate of
mutagenesis under each of the different conditions. To obtain
more detailed and quantitative data than would be possible using
a T7 assay, we analysed the samples by Sanger sequencing. First,
we plotted the mutation frequencies for all eight gRNAs infil-
trated at either OD600 = 1.5 or OD600 = 0.15 for each of the four
different plant variants, from this point forwards referred to as
epigenotypes (TRV-WT-infected, TRV-35S-infected, nonin-
fected and S1). In general, regardless of the epigenotype, higher
bacterial densities resulted in higher mutation frequencies
(P < 0.001, two-tailed t-test). However, differences in mutation
rate between the epigenotypes could be seen mainly with the low
bacterial density and we assumed that they were partially masked
when the plants were infiltrated with the higher bacterial density
(Fig. S2B). Interestingly, TRV-WT-infected plants exhibited
equally high mutation frequencies for both bacterial densities
(P = 0.575, two-tailed t-test) and the mutation frequencies for
the TRV-WT-infected plants were higher than in any of the
other plant variants, regardless of the bacterial density used for
delivering the CRISPR/Cas9 components. When comparing
noninfected and TRV-WT-infected 16c plants, which were infil-
trated at OD600 = 0.15, there was a significant difference in
mutation frequency (P = 0.001, two-tailed t-test). This suggests
that the TRV infection somehow predisposes the plant to a lower
fidelity of DNA repair, which we speculate could arise from the
plant’s stress response to the virus infection or even interference
of TRV RNA with the DNA repair pathways. This result high-
lighted the importance of our experimental controls to accurately
interpret our data. Due to the observed effect of TRV infection
on mutation frequency, it is only meaningful to make pairwise
comparisons between the TRV-WT-infected and TRV-35S-
infected plants or between the noninfected and S1 plants when
assessing the effect of chromatin state.
Based on the global analyses outlined above, we chose to anal-
yse the mutation frequencies for each gRNA individually for the
four different plant variants using the lower bacterial density
(OD600 = 0.15). To accurately discern the effects of promoter
methylation on the efficacy of CRISPR/Cas9-induced gene edit-
ing for each locus, we analysed the mutation frequency of the cor-
responding amplicons using TIDE-coupled Sanger sequencing
(Fig. S2C) and subsequently by Illumina NGS (Fig. 2c) using at
least three independent biological replicates. In agreement with
previous findings (Sentmanat et al., 2018), there was a strong cor-
relation between the Sanger and NGS datasets for all epigeno-
types and targets, although mutation frequencies determined by
NGS were, on average, 1.14 times higher than the estimates
determined by Sanger sequencing (Fig. S3). Comparing the
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Research 2291
TRV-WT-infected and TRV-35S-infected plants, the mutation
frequency was clearly higher for the promoter with active chro-
matin compared with the silenced, methylated promoter and this
trend was consistent across the majority of targets, with pb, mt, dt
and db targets showing statistically significant differences
(P < 0.05, one-tailed t-test; Figs 2c, S2C, left panels). Interest-
ingly, the difference in mutation frequency became significant for
ut when the number of biological repeats was increased from
three (NGS) to five (Sanger; Fig. S2C).
When we compared the mutation frequencies for gRNAs tar-
geted to either the top or bottom strand for a given locus, we found
no global correlation between the orientation (top or bottom) of
the target and the mutation frequency (data not shown). As the
impact of cytosine methylation on the mutation frequency in the
promoter region was significant, when comparing the nonmethy-
lated TRV-WT with heavily methylated TRV-35S plants
(P = 0.009, two-tailed t-test; Figs S5, S6), we were interested to also
assess the impact of cytosine methylation of transcribed DNA on
the efficacy of CRISPR/Cas9-mediated gene editing. To this end,
we induced RdDM at the coding region of GFP with a recombi-
nant TRV VIGS vector (TRV-GFP) and subsequently analysed the
frequency of Cas9-induced mutations by amplicon sequencing
(Notes S1; Fig. S4). We observed no statistically significant differ-
ence in mutation frequency between the TRV-GFP and TRV-WT
(control) samples for individual target sites (Fig. S4) and no statisti-
cally significant effect of the cytosine methylation on the mutation
frequency (P = 0.640, two-tailed t-test; Figs S5, S6), implying that
the TRV-GFP-induced cytosine methylation has little or no effect
on the frequency of CRISPR/Cas9-induced mutations for three
GFP targets including dtg (Figs 2b top panel, S4G,H, S5, S6).
Taken together, our data showed that the frequency of CRISPR/
Cas9-induced mutations at the promoter DNA was generally
inversely correlated with its level of cytosine methylation, but this
phenomenon did not apply to coding regions of DNA, suggesting
that the effect of DNA methylation on the efficacy of gene editing
was rather indirect (see the second paragraph of Discussion section).
The mutational outcome of CRISPR/Cas9 editing is
affected by DNA methylation
Next, we were interested to see if DNA cytosine methylation
could affect not only the frequency of CRISPR/Cas9-induced
mutations but also the mutational outcome. To this end, we used
the output of CRIPSRESSO2 software (Clement et al., 2019, p.
20) to analyse our NGS data to resolve the specific features of
insertion (IN), deletion (DEL) and substitution (SUB) events
within our dataset. SUB events occurred along the whole anal-
ysed DNA regions without any specific accumulations near the
predicted Cas9 cut sites, including negative controls that had
been infiltrated with a CRISPR/Cas9 cassette with no gRNA,
and accounted for c. 2% of reads. All these data indicated that
SUB events can be considered as sequencing or PCR errors,
therefore they were omitted from subsequent analyses.
Detailed analysis of total reads of IN and DEL events in indi-
vidual target sites showed that each target site had a specific pat-
tern of IN : DEL ratios. At some target sites IN dominated over
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DEL and vice versa (Figs 3, S7A). Interestingly, for three sites,
T AAA G A C T G G Sequence of the ut sgRNA targeted strand
the IN : DEL ratio was obviously shifted in connection with A T T T CTG

RdDM, either in favour of insertions (mt and db site) or deletions T A A A A G A C T G G 95% of A insertions
T A A A T G A C T G G 4% of T insertions
(dtg site; Fig. S7B). Moreover, we found that RdDM had a signif-

of 1bp insertion events

4th nt A T A G A G C T A
icant effect on the frequency of insertions and deletions at several
100%
individual target sites (pb and dt sites: Fig. 3a, left panel; and pb,

(NGS OD 0.15)
Frequency
80%
mt, dt, db and dtg sites: Fig. 3b, left panel). 60%
When we analysed IN mutations, c. 98% of them represented 40%
single-nucleotide insertions (Fig. S8) which were located just next 20%
0%
to the predicted Cas9 cut site. We observed a strong bias within
the inserted nucleotides towards the 4th nucleotide upstream of ut ub pt pb mt mb dt db dtg
the PAM at most target sites, which is consistent with a previous A C G T TRV
RV-WT TRV
RV-35S Non-infected S1
report from a nonplant model that Cas9 can produce either blunt Fig. 4 Detailed analysis of single-nucleotide insertion events detected next
ends or staggered ends with single-nucleotide (the 4th nucleotide to the Cas9 cut site. 1-bp insertion events were split into four categories,
one each for every nucleotide (A, C, G, T). The top bar shows which
from PAM) 50 overhangs (Figs 4, S9) (Li et al., 2015). The only
nucleotide was in the original sequence on the 4th position upstream from
exceptions were pb and mb, for which nontemplated T and A the protospacer adjacent motif (PAM) sequence. Each data point repre-
nucleotides were the most prevalent single-nucleotide insertions sents the mean of next-generation sequencing data (OD600 = 0.15) from
over the templated Gs (Figs 4, S9). Interestingly, the identity of three independent biological replicates. Terms TRV-WT, TRV-35S, nonin-
the inserted nucleotide also appeared to be affected by the fected and S1 are explained in Fig. 1; colour-coded CRISPR/Cas9 target
sites ut, ub, pt, pb, mt, mb, dt, db are explained in Fig. 2 and dtg in Sup-
epigenotype to varying degrees for different target loci, with the
porting Information Fig. S4.
biggest effect seen for the site targeted by the dt gRNA (Figs 4,
S9).

(a) Insertions

40%
ns ns ns
* ns ns
* ns ns
40%
ns ns ns ns ns ns ns ns ns
Percentage of reads
Percentage of reads

30% 30%
(NGS OD 0.15)
(NGS OD 0.15)

20% 20%

10% 10%

0% 0%
ut ub pt pb mt mb dt db dtg ut ub pt pb mt mb dt db dtg
TRV
RV-WT TRV
RV-35S Non-infected S1

(b) Deletions

40%
ns ns ns
* * ns
* * * 40%
ns ns ns ns ns ns ns ns ns
Percentage of reads
Percentage of reads

30% 30%
(NGS OD 0.15)
(NGS OD 0.15)

20% 20%

10% 10%

0% 0%
ut ub pt pb mt mb dt db dtg ut ub pt pb mt mb dt db dtg
TRV
RV-WT TRV
RV-35S Non-infected S1
Fig. 3 The impact of epigenotype on CRISPR/Cas9-induced mutations. (a) Frequencies of insertion and (b) deletion events at each target site and epigeno-
type. Individual points (o, x, □) represent next-generation sequencing data (OD600 = 0.15) from three independent biological replicates. Lower range, box,
upper range and a horizontal line represents the first quartile, second and third quartile, fourth quartile and median, respective. Two-way ANOVA was used
to examine the effect of the target position and epigenotype on the frequency of insertions and deletions and showed statistically significant interaction
between the target position and the epigenotypes (F(8, 36) = 5.097, P < 0.001 and F(8, 36) = 7.347, P < 0.001, respective) in infected plants. The interac-
tion was not significant for noninfected plants nor for insertions or deletions (F(8, 36) = 1.094, P = 0.390 or F(8, 36) = 1.087, P = 0.394). For both groups,
infected and noninfected, the simple and main effect of the target site was significant (P < 0.001). To examine the effect of the epigenotype in the individ-
ual target site a one-tailed t-test was used, as individual target sites had only two independent variables. * and ns indicate significant difference for P < 0.05
and not significant, respectively. Terms TRV-WT, TRV-35S, noninfected and S1 are explained in Fig. 1; colour-coded CRISPR/Cas9 target sites ut, ub, pt,
pb, mt, mb, dt, db are explained in Fig. 2 and dtg in Supporting Information Fig. S4.

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We then evaluated DEL events based on the length and position DNA ends with single-stranded overhangs in nondividing
of deletions relative to the predicted CRISPR/Cas9 cut site and the cells are prone to be repaired by the MMEJ pathway if an iden-
PAM. We found that only a small fraction of reads (3%) contained tical sequence of 2–25 nucleotides is present at both 30 DNA
mutations outside a 20-nt window around the Cas9-induced DSB ends near the cut site (Allen et al., 2019; Beying et al., 2021).
(Group 5; Fig. 5a). Close inspection of DEL events within this 20-nt DNA repair by this MMEJ mechanism results in a deletion (in
window resulted in the identification of four groups. DEL events in the simplest scenario that does not involve insertions) that
group 1 spanned the cut site in both a PAM-proximal and PAM- spans one of the two matching microhomology regions and the
distal direction. Group 2 contained DEL events that were exclusively sequence in between them. Consequently, the preserved copy
in the PAM-distal region adjacent to the Cas9 cut site. Group 3 con- of the microhomology region can be mapped to either side of
tained DEL events that could be mostly mapped on the PAM distal the deletion, and this mutational signature can be used to infer
end where terminal nucleotides can be mapped to either side of the likely cases of MMEJ-directed repair. In our dataset, we
Cas9 cut site. Finally, group 4 contained DEL events that were exclu- observed increased occurrence of such deletions only for ub and
sively in the PAM-proximal region adjacent to the Cas9 cut site dt targets, in which trinucleotides present at the terminus of the
(Fig. 5a,b). We found the distribution of mutations within each of PAM strand were repeated on the PAM-distal DNA end near
these four groups to be variable for the different gRNAs with no the Cas9 cut site (Fig. 6a). This indicated that the MMEJ
noticeable effect of the epigenotype. Groups 1, 2, 3 and 4 represented repair pathway did not play a significant role in our experimen-
1%, 61%, 27% and 7% of the total reads, respectively, indicating tal system, which is consistent with its predominant activity in
that most deletions occurred in the PAM-distal region adjacent to dividing cells. However, the surprisingly high occurrence of 1-
the Cas9 cut site (Figs 5c, S10). Intriguingly, we found that in many nt microhomologies in our dataset (Fig. 6a) implied that a sig-
cases, especially in group 2, the deletion was insulated from the pre- nificant number of the deletions were formed by a single-
dicted Cas9 cleavage site by at least one additional nucleotide, which nucleotide-dependent microhomology repair distinct from the
was almost exclusively aligned to the 4th nucleotide upstream of the classical MMEJ pathway. Moreover, when we analysed the 1-nt
PAM. This observation is consistent with Cas9-induced staggered microhomologies in more detail for individual gRNA targets,
ends with single-nucleotide 50 overhang (see Fig. 4), which mostly we noted that they nearly always aligned to the nucleotide that
did not vary between epigenotypes (Figs 5d, S11). was either 3rd or 4th nt upstream of the PAM (Figs 6b, S12).

(a) Distal Proximal

(c) 100%
1
(NGS OD 0.15)
Frequency

–20 +20 80%

2 –20 or +20
60%
40%
3 –20 +20 20%
4 –20 +20 0%

5 –20 +20
ut ub pt pb mt mb dt db dtg
TRV
RV-WT TRV
RV-35S Non-infected S1

(b) (d) G T A A A G A C T G G Sequence of the ut PAM strand

2
PAM CA T T T C TGA CC
GT A A A GAC T GGCG
G - - - - G A C T G G 63% of deletions only
GT A - - - - - T GGCG G - - - A G A C T G G 37% of deletions followed by remaining nt next to the cut site
1 GT AA - - - - - - - C G

GT - - - G A C T GG C G 4th nt A T A G A G C T A
G - - - - G A C T GG C G
2 GT - - A G A C T GG C G
100%
(NGS OD 0.15)
Frequency

G - - - A G A C T GG C G 80%
or
60%
G - - - - - A C T GGC G
3 GT A - - GAC T GGCG
40%
20%
GT AAA - - - - GGCG
4 GT AAA G - - T GGCG 0%
ut ub pt pb mt mb dt db dtg
del A C G T TRV
RV-WT TRV
RV-35S Non-infected S1
Fig. 5 Detailed analysis of deletion events detected next to the Cas9 cut site. (a) Schematic of mutation categories: (1) deletions on both DNA ends, (2)
only on the protospacer adjacent motif (PAM) distal end, (3) mostly on the PAM distal end where terminal nucleotides can be mapped to either side of
Cas9 cut DNA, (4) only on proximal DNA end, and (5) not directly adjacent to the cut site. (b) Schematic of mutations at nucleotide resolution for
categories 1–4 using representative sequences from the ut next-generation sequencing (NGS) data as an example. (c) Type and frequency of deletion
mutations in CRISPR/Cas9 cut sites. (d) Detailed analysis of group 2 and its first adjacent position next to the cut site on the PAM distal DNA end. Position
can contain plain deletion or a mutation with a remaining single nucleotide (A, C, G or T) adjacent to the predicted double-stranded break. The top bar rep-
resents which nucleotide was in the original sequence on the 4th position upstream from the PAM sequence. The top panel shows a detailed example of
‘ut’ gRNA target for a TRV-WT-infected sample. Each value represents the mean of NGS data (OD600 = 0.15) from three independent biological replicates.
Terms TRV-WT, TRV-35S, noninfected and S1 are explained in Fig. 1; colour-coded CRISPR/Cas9 target sites ut, ub, pt, pb, mt, mb, dt, db are explained in
Fig. 2 and dtg in Supporting Information Fig. S1.

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(a) (c) 5 nt DEL 3rd PAM

G G G T T G G 3c
3c A A C G A A A C T T C T G C A C C A A C C 5c

9 nt DEL 4th PAM

T T G G T T G G 3c

Normalised reads
3c G A A C G A A A C T T C T G C C C A A C C 5c

(NGS OD 0.15)
80%
60% 6%
db

(NGS OD 0.15)
40%

Frequency
20% 4%

0%
2%
0 1 2 3 4 5
Size of microhomology
il
e ta

surrounding DEL 0%
rohomology in d

on PAM DNA strand (nt)

Size of DEL (nt)
Mic

(b) A AGAC T GG GAC AGT GG C A T CGT GG GC A AGT GG C A AGGAGG

C T GT T AGG GGC AGAGG T GGGT GGG GT GGT T GG

AG TG AC GC A T GG CA TG AA
100%
(NGS OD 0.15)

80%
Frequency

60%
40%
20%
0%
ut ub pt pb mt mb dt db dtg
0 A C G T TRV
RV-WT TRV
RV-35S Non-infected S1
Fig. 6 Microhomology-mediated DNA repair between the protospacer adjacent motif (PAM)-proximal (PAM strand) and distal (non-PAM strand) DNA
ends can explain increased frequency in deletion patterns. (a) Analysis of the size of microhomologies surrounding deletion reads. Red lines represent the
probability of such microhomologies happening by chance, for each size class. As the largest detected microhomology was 5 nucleotides (nt) long, the
probability for no microhomology (indicated on chart as 0) was calculated as subtracting the sum of the calculated probabilities of the different microho-
mology sizes from 100%. For normalisation, the sum of all deletion reads in individual samples was normalised to 100% and further divided into individual
categories according to the microhomology size. The horizontal black line within the box, the ‘x’ and the diamond symbol represent the median, the mean
and the outliers, respectively. (b) Normalised frequency of deletion reads surrounded by no (0 nt) or 1 nt microhomology in individual target sites and
epigenotypes. The colour code indicates the occurrence of individual nucleotides. Sequences above each gRNA target represent the target sequence with
highlighted and colour-coded 3rd and 4th nucleotide upstream from PAM. Individual biological replicates can be found in Supporting Information Fig. S12.
(c) Enrichment of deletion sizes that match the distance between the nucleotide adjacent to the cut positions on the PAM strand and the corresponding
nucleotide position upstream from the cut site on the non-PAM-distal DNA strand in ‘db’ gRNA target. The example above the chart represents the enrich-
ment of 5 and 9 nt deletions, which correspond to the 3rd and 4th nucleotide microhomologies, respectively. Deletion patterns for the remaining target sites
can be found in Fig. S13. Each data point represents the mean of next-generation sequencing (NGS) data (OD600 = 0.15) from three independent biological
replicates. Terms TRV-WT, TRV-35S, noninfected and S1 are explained in Fig. 1; colour-coded CRISPR/Cas9 target sites ut, ub, pt, pb, mt, mb, dt, db are
explained in Fig. 2 and dtg in Fig. S4.

This again was consistent with our conclusion that Cas9 could
Discussion
cut either in front of the 3rd or at the 4th nt upstream of the
PAM on the PAM-containing strand. When we considered all The primary aim of this study was to investigate the effects of an
deletion mutations in our dataset, we observed an enrichment epigenetic modification, namely DNA cytosine methylation, on
of deletion sizes corresponding to the distance between the pre- the efficacy of generating targeted mutations using CRISPR/
dicted Cas9 blunt/staggered cleavage site and the position of a Cas9. Previous studies have suggested that the frequency of
single-nucleotide microhomology at the PAM-distal end of CRISPR/Cas9-induced mutations may be affected by the epige-
DSB (Figs 6c, S13). Together with the fact that the majority of netic status of the target DNA, using genomewide analyses to
DELs occurred on PAM-distal DNA strands (Figs 5c, S10, look for associations between the frequency of mutations and the
group 2) our data suggested that the nonrandom mutations epigenetic status (euchromatin vs heterochromatin) of the target,
were produced by a previously unidentified 50 DNA single- for multiple endogenous loci (Chen et al., 2016; Daer et al.,
nucleotide microhomology-mediated DNA repair. This high- 2017, 2018). To our knowledge, no study has to date compared
lighted the importance of considering the potential for trigger- the effect of DNA methylation on CRISPR/Cas9-induced muta-
ing single-nucleotide microhomology-mediated DNA repair tions by comparing the mutation frequency at the same locus
when designing gRNAs. containing either a high or low cytosine methylation content. In

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this work, we developed an ‘epigenetic switch’ that enabled us to
control the methylation status of the genomic loci, therefore
allowing us to compare the efficacy of CRISPR/Cas9-induced
mutations for a given locus containing either a high or low level
of cytosine methylation.
Our results showed that the level of cytosine methylation can
affect the efficacy of CRISPR/Cas9 mutagenesis in a target site-
specific manner (Figs 2c, S2C). The RdDM pathway, which we
used to trigger local chromatin modifications, is known to result
in transcriptional silencing if a promoter sequence is targeted
(Jones et al., 2001). By contrast, cytosine methylation introduced
to the transcribed region of a gene does not prevent transcription
and in some cases has even been reported to positively correlate
with high level transcription (To et al., 2015). As we did not
observe any change in the mutagenesis frequency in the tran-
scribed GFP coding region, which was heavily methylated in
TRV-GFP-infected plants (Fig. S4), we concluded that the
induced DNA methylation affects CRISPR/Cas9 mutagenesis
indirectly. In plants, cytosine methylation in the promoter region
can attract SUVH proteins and therefore induce histone H3K9
dimethylation (Johnson et al., 2007), which is associated with
repressive chromatin. In addition, methyl-CpG binding proteins
can attract histone deacetylases (Zemach & Grafi, 2003), which
increased positive charge density on the histone tails, allowing the
histones to wrap DNA more tightly. Methylated DNA can also
prevent the incorporation of the H2A histone variant H2A.Z,
which has been shown to affect the transcription of nearby genes
(Coleman-Derr & Zilberman, 2012). These and other associated
chromatin changes could make DNA less accessible for the
CRISPR/Cas9 machinery in the targeted P35S promoter. Indeed,
in mouse cells, Heterochromatin protein 1 (HP1)-associated
stable heterochromatin has been shown to reduce the ability of
the CRISPR/Cas9 complex to scan for potential target sites
(Knight et al., 2015), and a very recent study using proliferating
human cells also demonstrated that chromatin state influences
CRISPR/Cas9 mutagenesis (Schep et al., 2021). As DNA methy-
lation can affect nucleosome positioning in the promoter region
(Kelly et al., 2012; Zhong et al., 2021), we could also envision
that changes in the nucleosome occupancy of CRISPR/Cas9-
targeted loci would influence the efficacy of genome editing.
Indeed, it has been shown (both in vitro and in yeast cells) that
recognition of PAM sequences by CRISPR/Cas9 complexes is
impaired when the PAM is situated at the nucleosome core (Hinz
et al., 2015; Yarrington et al., 2018).
In contrast with our observation for the P35S promoter region,
ongoing transcription of the GFP gene can be associated with
repeated chromatin remodelling (Farnung et al., 2021) therefore
preventing methylation-induced changes in the chromatin struc-
ture. This would be in line with previous in vitro studies in
human cell cultures, for which target site methylation had no
impact on CRISPR/Cas9 activity (Hsu et al., 2013; Fujita et al.,
2016). Intriguingly, the higher mutagenesis rate that we found
within the GFP coding region in TRV-35S-infected plants com-
pared with TRV-GFP-infected plants (Fig. S4H) suggested that
active transcription may negatively interfere with CRISPR/Cas9
binding and/or cleavage, which was in agreement with previous
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Research 2295
findings (Daer et al., 2017). Alternatively, DSBs might be more
accurately repaired (resulting in lower rates of mutagenesis) by
the RNA-templated DSB repair mechanism, in which RNA
polymerase II transcripts can serve as the DNA repair template
(Keskin et al., 2014). Further experiments are required to deter-
mine which of the above mechanisms may underpin the effect of
DNA methylation on CRISPR/Cas9 activity in plants.
In addition to observing that induced cytosine methylation
could affect the rate of CRISPR/Cas9 mutagenesis, we noted that
several other factors within our experimental system also had a
significant effect (Fig. 7a). Virus infection generally increased the
frequency of mutagenesis (Figs 2c, S2C, left vs right panels). We
speculate that this effect was likely to be due to the stress caused
by the infection, which might have caused a higher error rate in
the cells’ DNA repair mechanisms. Indeed, a previous report has
linked heat stress to higher rates of CRISPR/Cas9 mutagenesis
(LeBlanc et al., 2018). Additionally, we observed that the density
of Agrobacterium used for infiltrating plants with the CRISPR/
Cas9 components had a significant effect on the rate of mutagen-
esis. The higher bacterial density of OD600 = 1.5 resulted in con-
sistently higher rates of mutagenesis compared with the lower
bacterial density of OD600 = 0.15 (Fig. S2B). From this, we con-
cluded that the level of expression of CRISPR/Cas9 components
or the number of cells successfully transformed within the infil-
trated patch was likely to be a limiting factor for the mutagenesis
frequency, over this range of OD600 values. Testing a wider range
of Agrobacterium densities would be required to determine the
optimum density of Agrobacterium suspension to achieve maxi-
mal mutagenesis frequencies. Importantly, our results highlight
the need to carefully determine the most appropriate Agrobac-
terium density to use for studies aimed at understanding specific
mechanisms of CRISPR/Cas9 editing. In our case, we chose to
use a lower bacterial density (OD600 = 0.15) for the subsequent
analyses, because the effect of cytosine methylation on CRISPR/
Cas9-induced mutagenesis frequency was partially masked when
a higher bacterial density was used (OD600 = 1.5; Fig. S2B). This
might be due to chromatin/histone changes that are related to
quickly repeating cycles of Cas9 cleavage.
As well as observing that extrinsic factors (virus infection and
bacterial density) significantly and consistently affected the rates
of mutagenesis for different target loci, we noted a strong intrin-
sic effect of the sequence itself for the different target loci
(Fig. 7a). In other words, some target loci appear to be more
prone to mutagenesis than others. Previous studies in human and
yeast cells have come to similar conclusions that the efficacy of
CRISPR/Cas9 mutagenesis is dependent on the sequence of the
target locus (van Overbeek et al., 2016; Lemos et al., 2018). It is
worth bearing in mind that the introduction of a mutation at a
target locus relies not only on Cas9 recognition and subsequent
DSB formation but also on the erroneous repair of that DSB.
The sequence of a target locus could potentially affect all three of
these steps (recognition, cleavage, and erroneous repair) that
underlie mutagenesis. Parsing these effects remains a significant
challenge to understanding how the underlying sequence of a tar-
get locus can predetermine the efficacy of CRISPR/Cas9 mutage-
nesis. Interestingly, even when we targeted the very same locus
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New
2296 Research Phytologist

(a) of staggered DSBs with single-nucleotide 50 overhangs that are

Variables
Epigenotype
Target CRIPSR/Cas9 Virus first filled in and then ligated presumably by c-NHEJ (Beying
Impact on site concentration infection
et al., 2021), which is the most prevalent DNA repair pathway in
Mutagenesis efficacy differentiated nonproliferating cells such as the leaf tissue in our
Ratio of blunt/staggered cuts study. We reviewed data from previous studies describing
Ratio of IN : DEL CRISPR/Cas9 mutations in several other plant species (including
Size of DEL
Arabidopsis, maize and cotton) and found that staggered-end for-
mation can also explain their most frequently inserted nucleotides
Size of IN
(Li et al., 2013, 2019; Nekrasov et al., 2013; Lee et al., 2019).
(b) These observations suggested that Cas9 can form both blunt and
3rd PAM sgRNA 4th PAM sgRNA staggered ends in planta. Interestingly, our data also revealed that
RuvC 3cW
DNA under RuvC 3c the frequency of blunt and staggered-end cuts is predetermined
Relaxed NGG
tension NGG
DNA
5c 5c
3rd
3c
5c
3c
5c
4th
3c
5c
by the CRISPR/Cas9 target site (Figs 4, 5, S9, S11), and also par-
3c
Target sequence HNH
5c
Target sequence HNH
tially influenced by the epigenotype (e.g. pt, dt and db). To our
knowledge, this is the first description of such an effect in any
CRISPR/Cas9 CRISPR/Cas9

Blunt cut Staggered cut

model system. To explain this phenomenon, we speculate that
the process of Cas9 unwinding the DNA duplex at a target locus
3rdN 4th
NGG
is sensitive to the physical tension of DNA in the locus, such that
Exo
Exo G GG Exo A G

5c
T GT C T T GAGCGGC A T T T
3c
5c
3c
T GT C T T GAGCGGC A T T T
3c
5c
relaxed DNA duplexes are more easily unwound than DNA
5c
3c
3rd 3rd
under tension. The tensioned conformation of DNA could there-
Fig. 7 Overview of CRISPR/Cas9 mutagenesis outcomes. (a) A table fore affect the position of the RuvC nuclease domain of Cas9 rel-
summarising the main conclusions of our study. Checkmarks and crosses ative to the nontargeted strand, causing a shift of the cut by one
indicate statistically significant and nonsignificant effects, respectively.
Mixed marks designate statistical significance for some, but not all target
nucleotide further from the PAM and resulting in a staggered
sites. (b) Proposed model for how DNA tension may influence the DSB (Fig. 7b). Therefore, the relaxed DNA might favour the for-
outcome of Cas9-induced double-stranded breaks (DSBs) to produce mation of blunt DSBs, whereas DNA under tension may favour
either a blunt or a staggered DSB and 50 single-nucleotide mediated DNA the formation of a staggered DSB. In agreement with our model,
repair. On relaxed DNA, Cas9 forms a blunt DSB three nucleotides Cas9 in vitro assays with DNA in a relaxed conformation pro-
upstream from protospacer adjacent motif (PAM) due to the RuvC and
HNH domains of Cas9 cleaving between the 3rd and 4th nucleotides
duced blunt cuts (Sternberg et al., 2014), whereas when we anal-
upstream from the PAM position on the PAM strand and non-PAM strand, ysed data from in vivo studies, for which DNA is more likely to
respectively (left panel). DNA under tension could stretch the PAM strand, be under tension at certain loci, we inferred that Cas9 frequently
causing the RuvC domain of Cas9 to cleave between the 4th and 5th produces staggered DSBs (Li et al., 2013, 2019; Nekrasov et al.,
nucleotides upstream of the PAM. This, together with HNH-mediated 2013; Lee et al., 2019). DNA tension could be affected by the
cleavage between the 3rd and 4th nucleotides upstream of the PAM posi-
tion on the non-PAM strand would result in a staggered DSB with a 1-nt 50
local chromatin environment, mainly by DNA-interacting pro-
overhang on the PAM strand (right panel). After cleavage by Cas9, the teins such as histones, transcription factors and polymerases.
PAM-distal DNA strand is probably released first because most of the dele- Their binding to DNA can be affected (or even directed) by
tions happened on the PAM-distal DNA (see Fig. 5). Moreover, the cytosine methylation, which could result in a highly localised and
nucleotide adjacent to the cut on the PAM-containing proximal strand strand-specific increase or decrease in DNA tension.
serves as a single-nucleotide searching for complementarity to allow speci-
fic 50 microhomology-mediated DNA repair (bottom panels).
In our system, we also observed that the epigenetic status of
gRNA target sites could influence the frequency of CRISPR/
Cas9-induced deletions (Fig. 3b). Interestingly, the vast majority
but on complementary DNA strands, we observed differences in of deletion events happened on the PAM-distal DNA end
the rates of mutagenesis. This effect was most visible for the mt/ (Figs 5c, S10, group 2). We also found similar deletion patterns
mb positions in all epigenotypes. The mutation frequency in other plant studies (Li et al., 2013, 2019; Nekrasov et al.,
induced by the mt gRNA in TRV-WT-infected plants was 2013; Kumar et al., 2018; Lee et al., 2019; Fellenberg et al.,
approximately four times higher than that of the mb gRNA, 2020), but not in animal model systems (van Overbeek et al.,
which targeted the same locus on the complementary strand 2016). Based on these findings, we assumed that in plants the
(Figs 2c, S2C). PAM-distal cleavage product is released first from the CRISPR/
According to the original and widely accepted model, Cas9 Cas9 complex, and not just the nontargeted PAM-distal DNA
nuclease produces blunt-ended DSBs (Jinek et al., 2012; Ahmad strand, as suggested by an animal in vitro study (Richardson
et al., 2020). More recent studies on animal and yeast models et al., 2016). In such a release mechanism, the PAM-distal DNA
showed that, in vivo, the Cas9 endonuclease can also form stag- end is more exposed to exonucleases, leading to deletions occur-
gered DSBs with single-nucleotide overhangs (Li et al., 2015; ring predominantly at this side of the cut, whereas the PAM-
Lemos et al., 2018). These results were also supported by molecu- proximal DNA end remains protected and mostly unchanged
lar dynamics simulations (Zuo & Liu, 2016). Our analyses of (Fig. 7b). Moreover, our analysis suggests that a significant pro-
insertion and deletion events clearly showed that a high propor- portion of such deletions may be directed by a 50 single
tion of these mutations can be most simply explained as repairs nucleotide at the PAM-proximal end of the Cas9-induced DSB

New Phytologist (2022) 235: 2285–2299 Ó 2022 The Authors

www.newphytologist.com New Phytologist Ó 2022 New Phytologist Foundation

New
Phytologist
(Figs 7b, S13), which we also observed in other plant data sets
(data not shown). Intriguingly, the well characterised
microhomology-dependent DNA repair pathway (MMEJ/alt-EJ)
is associated with 30 DNA overhangs and proliferating cells (Sfeir
& Symington, 2015). Therefore, it is plausible that the 50 single-
nucleotide microhomology-mediated DNA repair that we
described in this study may be governed by NHEJ or novel
genetic factors. However, more data including genetic evidence is
required to identify the underpinning molecular mechanism and
consequently assign the corresponding DNA repair pathway.
Taken together, our findings (Fig. 7a) have demonstrated that
DNA methylation can influence the mutation frequency and
mutational outcomes in the context of CRISPR/Cas9 genome
editing, probably indirectly through changes in chromatin state.
Our findings including the Cas9 staggered-end-mediated inser-
tions and deletions and 50 DNA single-nucleotide mediated DNA
repair (Fig. 7b) may contribute to better target selection and gRNA
designs for diverse future applications in plants and beyond.
Acknowledgements
We are grateful to Professor David Baulcombe for 16c N. ben-
thamiana seeds, Dr Holger Puchta for pEn_Chimera and
pDe_CAS9 plasmids, Muriel Monteiro and Aron Ferenczi for
fruitful discussions, and to Professor Andrew Hudson for critically
reading the manuscript. This work was supported by the Royal
Society International Collaboration Award IC170320, Wellcome
grant (206194) and AP was supported by the Charles University
project GA UK (1212218), the STSM Grant from COST Action
(CA15223) and by Nadacnı fond Prırodovedecke fakulty UK.
Competing interests
None declared.
Author contributions
AM performed conceptualisation; AM and AP performed
methodology; AP performed software; AP, AM, LF and AB
investigated the research; AP, LF and AM wrote original draft;
DEP and AB reviewed and edited; AP visualised; and AM super-
vised the study.
ORCID
Andrew Bassett https://orcid.org/0000-0003-1632-9137
Lukas Fischer https://orcid.org/0000-0003-1852-0461
Attila Molnar https://orcid.org/0000-0002-1044-6327
Adela Pribylova https://orcid.org/0000-0001-8675-5065
Douglas E. Pyott https://orcid.org/0000-0001-6340-9266
Data availability
The NGS data have been deposited in the ArrayExpress database
at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession
number E-MTAB-10256.
Ó 2022 The Authors
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Research 2297
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Supporting Information
Additional Supporting Information may be found online in the
Supporting Information section at the end of the article.
Fig. S1 GFP fluorescence phenotypes for individual epigenotypes
before agroinfiltration.
Fig. S2 Analysis of mutagenesis frequency at CRISPR/Cas9 cut sites.
Ó 2022 The Authors
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Fig. S3 Comparison of the overall frequency of mutations
induced by CRISPR/Cas9 in P35S.
Fig. S4 The impact of gene-body methylation on CRISPR/Cas9-
induced mutations.
Fig. S5 Analysis of DNA (cytosine) methylation at CRISPR/
Cas9 gRNA target sites in individual epigenotypes.
Fig. S6 Correlation analysis between the mutation frequencies
and the percentage of methylated cytosines in the target locus in
the compared epigenotypes.
Fig. S7 Comparison of insertion (IN) and deletion (DEL) events
in individual CRISPR/Cas9 target sites.
Fig. S8 Insertion events analysis in P35S and GFP coding region.
Fig. S9 Detailed analysis of single‑nucleotide insertion events
detected next to the Cas9 cut site in individual epigenotypes.
Fig. S10 Detailed analysis of the type and frequency of deletion
mutations in individual epigenotypes.
See also the Commentary on this article by Raffan et al., 235: 2146–2148.
Ó 2022 The Authors
New Phytologist Ó 2022 New Phytologist Foundation
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Research 2299
Fig. S11 Detailed analysis of group 2 and its first adjacent posi-
tion next to the cut site on the PAM-distal DNA end in individ-
ual epigenotypes.
Fig. S12 Detailed analysis of the normalised frequency of dele-
tion reads surrounded by no (0 nt) or 1 nucleotide microhomol-
ogy in individual target sites and epigenotypes.
Fig. S13 The pattern of the deletion sizes in individual target
sites.
Notes S1 DNA cytosine methylation within coding sequences
does not alter the frequency of CRISPR/Cas9-induced mutations.
Table S1 List of primers used in this study.
Table S2 List of vectors used in this study.
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