The CRISPR Journal

Volume 7, Number 6, 2024

ª Mary Ann Liebert, Inc.
DOI: 10.1089/crispr.2024.0003

RESEARCH ARTICLE

Mutation-Specific CRISPR Targeting with SaCas9

and AsCas12a Restores Therapeutic Sensitivity
in Treatment-Resistant Melanoma
Brett M. Sansbury,1 Sophia B. Masciarelli,1 Salma Kaouser,1 Olivia M. Tharp,1,2 Kelly H. Banas,1 and Eric B. Kmiec1,*

Abstract
Background: Melanoma remains one of the most challenging cancers to treat effectively with drug resistant
remaining a constant concern, primarily with activating BRAF mutations. Mutations in the BRAF gene appear
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in approximately 50% of patients, 90% of which are V600E. Two frontline BRAF inhibitors (BRAFi), vemurafenib
and dabrafenib, are frequently used to treat unresectable or metastatic BRAF V600E melanoma. Initial
response rates are high, but soon thereafter, 70–80% of patients develop resistance to treatment within a
year. A major mechanism of resistance is the generation of a secondary Q61K mutation in the NRAS gene.
Methods: We have developed an approach in which a CRISPR-Cas complex can be designed to distinguish
between mutant genes enabling resistance to standard care in tumor cells and normal genomes of healthy
cells. For the first time, we demonstrated the utility of two CRISPR-directed mutation-specific editing
approaches to restore BRAFi sensitivity in BRAFV600E/NRASQ61K resistant A375 cells.
Results: We utilize an AsCas12a protospacer adjacent motif site created by the NRAS Q61K mutation and the
Q61K mutation in the critical seed region of an SaCas9 sgRNA for Q61K-selective targeting. We show here
that both approaches allow for effective NRAS targeting of only mutated-Q61K and after CRISPR-directed
Q61K-targeting, previously resistant A375 cells are re-sensitized to BRAFi treatment.
Conclusion: Our data support the feasibility of the development of CRISPR-Cas therapeutic approaches to
the treatment of melanoma. Successful therapeutic CRISPR-directed gene editing would enable both specific
and efficient editing of a mutation-specific targeting approach eliminate concern for on- and off-target dam-
age to the genomes of healthy cells.

Introduction response rates and improved progression-free survival,

In the United States, skin cancer is the most common form 70–80% of patients still develop resistance within 1 year.8,9
of cancer, with melanoma being the most lethal form.1 One mechanism of resistance is the generation of secondary
Activating v-raf murine sarcoma viral oncogene homolog mutations in the neuroblastoma RAS viral oncogene homo-
B1 (BRAF) mutations are seen in approximately 50% of log (NRAS) gene where a C > A base substitution results in
melanomas, and 90% of these mutations are V600E.2,3 a change of the amino acid in position 61 from glutamine
Two frontline BRAF inhibitors (BRAFi), vemurafenib and to lysine (Q61K).2,10 Once developed, mutant NRAS will
dabrafenib, are frequently used to treat unresectable or met- preferentially bind with CRAF and reactivate Ras-Raf-
astatic BRAF V600E melanoma.4,5 Acquired resistance is MEK-ERK (MAPK) pathway (Fig. 1).11 This secondary
well known and remains a challenging obstacle to long- mutation not only renders treatment ineffective, but once
term treatment efficacy.2,6 It has been suggested that a strat- the NRAS Q61K mutation is acquired, the expression of
egy often used to mitigate such outcomes is combinatorial genes involved in the epithelial-to-mesenchymal transition
treatments involving two or more drugs,7 although combi- paradoxically promote invasiveness and metastasis is dra-
natorial BRAF and MEK inhibition does show initial high matically enhanced.12

1
Gene Editing Institute, ChristianaCare Health System, Newark, Delaware; and 2Department of Medical and Molecular Sciences, University of Delaware, Newark, Delaware.

*Address correspondence to: Eric B. Kmiec, Gene Editing Institute, 550 South College Ave, Suite 100A, 2nd Floor, Newark, Delaware 19713, E-mail: Eric.B.Kmiec@
christianacare.org

366
 CRISPR TARGETING TO SENSITIZES RESISTANT MELANOMA 367

FIG. 1. Mechanism of BRAFi resistance through an acquired NRAS Q61K mutation. The Ras-Raf-MEK-ERK
pathway is shown as influenced by BRAF/NRAS mutational status, noted as wild-type (WT) or mutated (V600E or
Q61K). The influence of BRAFi treatment on BRAFV600E/NRASWT is shown inhibiting the MAPK pathway while
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BRAFV600E/NRASQ61K status shows the mechanism of inhibition escape and MAPK overexpression. Restored
sensitivity to BRAFi treatment is shown after NRAS Q61K MT-selective editing.

Advancements in gene editing technologies over the the sgRNA are detrimental to DNA cleavage activity for
past decade have opened doors to its use in genetic medi- SaCas9.28,29 For the NRAS Q61K mutation, an SaCas9-
cine and targeted therapies. The therapeutic potential of targetable site is available at that location that generates an
CRISPR gene editing has been spotlighted for diseases opportunistic mismatch in PAM proximal position 4 in the
such as Sickle Cell Disease, Duchenne muscular dystro- sgRNA, well within the critical seed region for selective
phy, Leber congenital amaurosis, and Hereditary trans- targeting (Fig. 2A and B). As a contrast, we include a non-
thyretin amyloidosis13–16 but concerns are still present selective SpCas9 target that cuts upstream from the Q61K
about the effects of CRISPR-induced collateral damage, mutation site, which indiscriminately edits both mutant
both on- and off-target.17–19 Additional gene editing tools and wild-type NRAS. We show here that both selective tar-
such as base and prime editors have been developed in geting approaches allow for effective and specific NRAS
attempts to increase specificity and decrease the chances targeting of only mutated-Q61K, while the wild-type Q61
of adverse, unintended outcomes from occurring.20–24 remain untargeted. Importantly, after CRISPR-directed
However, their overall effectiveness and universality have Q61K-targeting, the previously resistant A375 cells are re-
been underwhelming and recently the genotoxic effects sensitized to BRAFi treatment as a direct result of editing
of these tools have emerged as serious challenges.25 The the Q61K mutation in both cases.
ultimate goal of any successful gene editing centers on
specific and efficient editing, and to this end mutation- Materials and Methods
specific targeting approaches would eliminate some con- Cell Culture Conditions. Parental and Isogenic A375
cern for on-target editing of healthy, normal cells.26,27 cell lines (ATCC, Manassas, Virginia) were cultured in
Here, we demonstrate the utility of two CRISPR- DMEM (Corning, Edison, New Jersey) supplemented with
directed mutation-specific targeting approaches to restore 10% FBS. Cells were incubated at 37C with 5% CO2.
BRAFi sensitivity in BRAFV600E/NRASQ61K-mutated, Transfection Conditions. Isogenic A375 cells (ATCC,
BRAFi-resistant A375 cells. Our first approach involves Manassas, Virginia) were split 24 h prior to nucleofection.
utilizing an AsCas12a protospacer adjacent motif (PAM) A 1e6 cell, 100ul reaction was set up using SF nucleofec-
site created by the NRAS Q61K mutation, allowing for tion solution (Lonza, Morrisville, NC) with pre-complexed
Q61K-selective targeting. Cas12a recognizes a PAM site RNPs at 1250:250 pmol ratio (gRNA:Cas protein). AsC
of 5’-TTTN-3’, and the Q61K mutation involves a CAA as12a (Ultra), SpCas9 (V3) nucleases, and their respective
to AAA nucleotide change which generates, on the com- crRNA and sgRNA were obtained from IDT (Coralville,
plementary strand, a new 5’-TTTC-3’ PAM site from the Iowa). SaCas9 nuclease and sgRNAs were obtained from
previous Q61 5’-TGTC-3’ site (Fig. 2A and B). Our sec- Synthego (Redwood City, CA). Specific gRNA sequences
ond strategy utilizes the Q61K mutation in the critical can be found in Supplementary Table S1. Nucleofection
seed region of an SaCas9 sgRNA for Q61K-selective tar- was performed using the FF-120 program on a 4D Nucle-
geting. Mismatches in PAM proximal positions 1–6 within ofector (Lonza, Morrisville, NC). After nucleofection,
 368 SANSBURY ET AL.

sequences. Indexing PCR was done using KAPA HiFi

HotStart ReadyMix PCR Kit (Roche Diagnostics, Florham
Park, NJ), cleaned up using AMPure XP beads (Beckman
Coulter, Brea, CA) and sequenced using a MiSeq System
(Illumina, San Diego, CA) with readouts visualized using
CRISPResso2.30
Sanger Sequencing PCR was done using BigDye Termi-
nator v3.1 (Thermo Fischer Scientific, Waltham, Massa-
chusetts) following the STeP-Up protocol.31 The resulting
DNA was purified using the ZR DNA-sequencing clean-
up kit (Zymo Research, Irvine, California) and sequenced
using a SeqStudio Genetic Analyzer (Thermo Fisher, Wal-
tham, Massachusetts) with readouts analyzed visualized
using DECODR.32
BRAFi Treatment and Cell Viability. BRAFi treat-
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ments were performed in experimental triplicate for each

condition. Cells were seeded at a 5,000 cell/well density in
a 96-well opaque, white plate. Cells were dosed at 100 nM
FIG. 2. NRAS Q61K mutation site and selective or 500 nM concentrations for dabrafenib or vemurafenib,
targeting. (A) The NRAS Q61K mutation site is shown respectively, as determined by dose-response for each drug
within a red box and a representation of the allelic shown in Supplementary Figure S1 at concentrations indi-
contributions of BRAFV600E/NRASQ61K A375 cell line is cated (Selleck Chemicals, Houston, Texas). Cells were
shown here, with 66% WT and 33% MT contributions. then incubated at 37C with 5% CO2 for 72 h. Cellular via-
(B) Two NRAS Q61K-selective CRISPR target sites are bility was done after 72 h using Cell Titer Glo 2.0 (Prom-
illustrated here, with the Cas12a (nPAM) PAM site ega, Madison, Wisconsin) on a TECAN Infinite® 200
shown in red on the 5’ end relative to the spacer PRO (Tecan, Mannedorf, Switzerland). Cell viability was
shown in orange and the SaCas9 (sMT) PAM site in red
determined by the viability of the targeted populations with
BRAFi treatment relative to the viability of same targeted
on the 3’ end relative to the spacer shown in green.
population without BRAFi treatment to control for the
PAM, protospacer adjacent motif; WT, wild-type.
potential effect of CRISPR editing on cell viability.

cells were plated in a 6-well plate with pre-warmed media Results

and allowed to recover for 72 h. To investigate the mutation-specific targeting selectivity
DNA Isolation, PCR Amplification, and Sequenc- of the Cas12a PAM site (nPAM) and the SaCas9 sgRNA
ing. Genomic DNA from cell pellets was extracted using seed region mutation (sMT) generated by the NRAS
a DNeasy Blood and Tissue kit (Qiagen, Hilden, Ger- Q61K mutation, we utilized ribonucleoprotein (RNP)
many). Genomic DNA was amplified using Phusion delivery of CRISPR molecules into isogenic A375 cells
High Fidelity PCR Master Mix with HF Buffer (Thermo containing both the primary BRAF V600E mutation and
Fisher, Waltham, Massachusetts) with parameters opti- the secondary, BRAFi resistance-inducing NRAS Q61K
mized for an amplicon size of 397 bp using PCR primers mutation (Supplementary Figure S1). This cell model
(Integrated DNA Technologies, Coralville, Iowa) FWD contains three copies of the NRAS gene, two wild-type
5¢-GACAAACCAGATAGGCAGA-3¢ and REV 5¢-CTA and one Q61K-mutated, resulting in an approximate
GTGTGGTAACCTCATTTC-3¢. PCR amplicons were allelic profile of 66% and 33%, respectively (Fig. 2A).
purified using the Select-a-Size DNA Clean and Concen- Targeting reactions were conducted by nucleofection of
trator kit (Zymo Research, Irvine, California) and verified preassembled RNP molecules and the editing efficiency
on a FlashGel System using 2.2% DNA Cassettes (Lonza, was determined via NGS on genomic DNA (post-target-
Morrisville, NC). Genomic analysis was performed by either ing). Sequencing showed that selective Q61K targeting
Next Generation Sequencing (NGS) or Sanger sequencing. with the Cas12a PAM dropped the mutated allele from
NGS Amplicon PCR, purification, and visualization approximately 33% to 15%, while the 66% wild-type con-
was done using as described previously, but with primers tribution remained unchanged (Fig. 3). Additionally, mock,
modified to include Illumina’s overhang adapter and non-targeting RNP (scrambled) transfection complexes
 CRISPR TARGETING TO SENSITIZES RESISTANT MELANOMA 369

FIG. 3. Genomic analysis of Q61K-selective Cas12a nPAM targeting. (A) NGS analysis is shown for Q61K-
selective Cas12a nPAM targeting reaction. The percentage of Q61K contribution remaining after targeting are
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shown as bold text. (B) A bar graph is shown representing the percentage of sequencing contributions after
editing for Q61 wild-type NRAS (green), Q61K mutated NRAS (red) and indels (gray). PAM, protospacer adjacent
motif.

showed no activity, no difference from the untreated con- position proximal to the SaCas9 PAM site, within the crit-
trols. These results confirm the mutation-specific cleavage ical seed region (Fig. 2B). WT-selective and MT-selective
selectivity of the nPAM Cas12a targeting approach. targeting with SaCas9 showed high editing efficiency and
Having shown that PAM creating mutations are selec- selectivity, with the wild-type contribution dropping from
tively targetable with Cas12a, we next examined the approximately 66% to 6% and Q61K-mutated contribution
selectivity of a seed region mismatch mutation that create from approximately 33% to 6%, respectively. For both
a unique, selective cleavage opportunity for SaCas9. Two WT- and MT-selective SaCas9 targeting reactions, the
selective sgRNAs were designed to target either wild- non-targeted contribution remained unchanged, confirming
type Q61 (WT-selective) or mutated Q61K (MT-selec- the selectivity of the sMT SaCas9 targeting approach
tive), with the mutation generating a mismatch in the 4th (Fig. 4A and B). Additionally, we were able to confirm

FIG. 4. Genomic analysis of WT- and MT-selective SaCas9 sMT targeting. (A) NGS analysis is shown for WT-selective
(top) and MT-selective (bottom) SaCas9 sMT targeting reactions. The percentages of Q61 wild-type and Q61K
contributions remaining after targeting are shown as bold text. (B) A bar graph is shown representing the percentage
of sequencing contributions after editing for Q61 wild-type NRAS (green), Q61K mutated NRAS (red), and indels (gray).
 370 SANSBURY ET AL.

WT- and MT-selective SpCas9 targeting activity when a for SpCas9 (Fig. 6C and Supplementary Figure S2). NGS
seed region mismatch was present in a separately targeted sequencing of the targeted populations shows a marked
loci, confirming sMT selective targeting in that additional reduction in the 33% mutant allele profiles across targeting
Cas9 ortholog (Data not shown). strategies. For both SaCas9 sMT and Cas12a nPAM selec-
Next, we examined how these Q61K-selective targeting tive targeting, with only 4.58% and 6.68% of the original
approaches impact functional NRAS activity with the aim 33% Q61K contributions remaining, respectively. For the
of restoring sensitivity to BRAFi-resistant A375 melanoma non-selective SpCas9 targeting, 2.12% of the Q61K contri-
cells. We selectively targeted Q61K mutant NRAS and, bution remains after targeting, but at the expense of indis-
after 72 h of recovery, the targeted populations were sub- criminate wild-type allele editing which also reduced the
ject to simultaneous genomic and functional analysis via wild-type profile from 66% to 7.64%.
NGS and BRAFi sensitivity as measured by cell viability
(Fig. 5). For this experiment, we used both SaCas9 sMT Discussion
and Cas12a nPAM Q61K-selective RNPs and an addi- CRISPR gene editing has emerged as a powerful tool
tional non-selective SpCas9 RNP to assess the therape- with therapeutic potential for the treatment not only of
utic effect of selective and non-selective disruption of the genetic diseases, but also of cancer, cardiovascular dis-
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Q61/Q61K target (Fig. 6A). After targeting and recovery, ease, chronic infection, and more.13–16,33–38 We present
measurement of cell viability revealed that both selective here two mutation-specific targeting approaches that bypass
Q61K approaches and non-selective targeting reactions the on-target editing concerns for healthy, non-mutated cells
successfully re-established sensitivity to previously resist- by targeting mutation-specific sequences, while also decre-
ance isogenic A375 cells. Cell survival for the edited popu- asing the number of potential off-target sites by utilizing
lations treated with Dabrafenib and Vemurafenib were Cas nucleases that require longer, more complex PAM sites
44% and 70% for SaCas9, 44% and 71% for Cas12a, and for efficient DNA binding and cleavage to initiate gene
31% and 61% for SpCas9, respectively (Fig. 6B). The editing.
genomic analysis additionally showed that the phenotypic Mutations in the BRAF gene are frequently seen in
response seen after BRAFi treatment was a result of the tar- patients with melanoma, with the V600E mutation being
geted editing introducing insertions and deletions (indels) most prominent.2,3 BRAFi, Dabrafenib and Vemurafenib,
which reduce the functional Q61K contribution either selec- are a very effective first-line therapy for BRAF-mutated
tively, for Cas12a or SaCas9 targeting, or non-selectively, melanoma but resistance to this treatment is usually seen

FIG. 5. Experimental design for BRAFi sensitivity and genomic analysis after Q61K-selective targeting. Q61K-
selective targeting via nucleofection was performed, followed by 72 h of recovery. Targeted populations were then
simultaneously subject to genomic analysis via NGS and BRAFi sensitivity assessment via treatment and cell viability.
 CRISPR TARGETING TO SENSITIZES RESISTANT MELANOMA 371

FIG. 6. Genomic analysis and BRAFi sensitivity after Q61K-selective targeting. (A) Selective (SaCas9 sMT and
Cas12a nPAM) and non-selective (SpCas9) targets are shown at the Q61K mutation site. (B) Cell viability data in
response to BRAFi treatment is shown for selective and non-selective targeting reactions, compared with
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Parental (BRAFi-sensitive) and Isogenic (BRAFi-resistant) A375 cells and transfection controls. (C) NGS sequencing
is shown of selective (SaCas9 sMT and Cas12a nPAM) and non-selective (SpCas9) targeted populations. The
percentage of Q61K contribution remaining after targeting are shown as bold text. (D) A bar graph is shown
representing the percentage of sequencing contributions after editing for Q61 wild-type NRAS (green), Q61K
mutated NRAS (red), and indels (gray).

within a year, even when used in combination with MEK non-selective SpCas9 targeting showed overall editing of
inhibitors.2,6,9 One mechanism of resistance to BRAFi approximately 90.2%, with 30.88% of the 33% Q61K
treatment is an acquired secondary Q61K mutation in the contribution disrupted and 58.36% of the 66% wild-type
NRAS gene (Fig. 1). Alternative treatment options are contribution also having been edited. In terms of BRAFi
limited once drug resistance has developed, emphasizing sensitivity after targeting, the selective nPAM and sMT
a need to develop new approaches to treat patients with targeting approaches showed comparable results to the
acquired BRAFi resistance. We present here the specific- non-selective targeting with both dabrafenib and vemura-
ity and efficiency of two Q61K-selective CRISPR target- fenib treatments as a direct result of the Q61K editing
ing approaches to restore sensitivity to treatment in a lowering the mutant contribution and thus re-sensitizing
BRAFi-resistant A375 cell line (BRAFV600E/NRASQ61K) cells to BRAFi treatment.
model for melanoma. We were able to selectively disrupt These results confirmed the selectivity of our nPAM
the Q61K mutation using two unique mutation-specific and sMT targeting approaches and confirmed the re-
targeting approaches, a new Cas12a PAM site generated sensitization of targeted populations to BRAFi is a result
by the Q61K mutation (nPAM) and an SaCas9 sgRNA of a reduced Q61K contribution, showcasing the effects
mismatch the Q61K mutation creates in the critical seed of selectively targeting Q61K while leaving the wild-
region (sMT) (Fig. 2A and B). type contribution unchanged is a preferable approach
NGS analysis of Cas12a nPAM and SaCas9 sMT when compared to the collateral damage seen with the
(MT- and WT-selecting sgRNAs) showed both strategies non-selective SpCas9 disruption of wild-type Q61. In
were efficient in their ability to disrupt the targeted addition to high specificity and efficiency for mutation-
allelic contributions and highly selective with no unin- specific targeting, in silico prediction done by Cas-OFF-
tended on-site targeting occurring (Figs. 3 and 4). We inder39 analysis for both SaCas9 and Cas12a shows a
also show that after selective (Cas12a, SaCas9) and non- markedly reduced number of off-target sites due to the
selected (SpCas9) targeting, disruption of functional increased complexity and length of the PAMs required
Q61K seen via NGS analysis reflects phenotypic changes for effective binding and cleavage of these Cas nucleases
seen in the targeted populations as re-sensitization to as compared to the non-selective SpCas9 described previ-
BRAFi (Figs. 5 and 6). The Q61K-selective targeting ously (Supplementary Figure S3). To firmly establish the
with Cas12a nPAM and SaCas9 sMT showed editing therapeutic value of this approach, we intend to character-
efficiencies of approximately 26.3% and 28.42% of the ize individual subpopulations of clones and measure the
33% Q61K contribution, respectively, with the 66% contribution of individual indel profiles. We have pre-
wild-type Q61 contributions remaining unchanged. The viously isolated clonal populations from targeted cells,
 372 SANSBURY ET AL.

and while not affirmative, the results provide valuable 4. Ascierto PA, Minor D, Ribas A, et al. Phase II trial (BREAK-2) of the BRAF
inhibitor dabrafenib (GSK2118436) in patients with metastatic mela-
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for tumor genomes will ultimately limit concerns of unin- 8. Jenkins RW, Sullivan RJ. NRAS mutant melanoma: An overview for the
clinician for melanoma management. Melanoma Manag 2016;3(1):
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Acknowledgment logical behavior and future strategies for therapeutic management.
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by members of the Kmiec research group at the Gene by NRAS mutants capable of promoting melanoma initiation. Nat Com-
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Editing Institute. 12. Chhabra G, Ahmad N. BRAF inhibitors in melanoma management:
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13. ClinicalTrials.Gov. Study to evaluate safety, tolerability, pharmacoki-
B.M.S.: Conceptualization, methodology, validation, visu- netics, and pharmacodynamics of NTLA-2001 in patients with hereditary
alization, formal analysis, project administration, writing— transthyretin amyloidosis with polyneuropathy (ATTRv-PN) and patients
original draft, review and editing; S.B.M.: Methodology, with transthyretin amyloidosis-related cardiomyopathy (ATTR-CM) - full
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investigation, writing—original draft; S.K.: Methodology, NCT04601051 [Last accessed: September 17, 2023].
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Disclosure Statement 16. ClinicalTrials.Gov. A study evaluating the safety and efficacy of EDIT-301
No competing financial interests exist. in participants with severe sickle cell disease (RUBY) - full text view - n.d.
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The research was supported by funds from the Lisa Dean challenges of the use of CRISPR/Cas9 as a replacement for cancer ther-
apy. Mol Cancer 2022;21(1):64; doi: 10.1186/S12943-021-01487-4
Moseley Foundation and the Ammon Foundation. 18. Höijer I, Emmanouilidou A, Östlund R, et al. CRISPR-Cas9 induces large struc-
tural variants at on-target and off-target sites in vivo that segregate across
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Supplementary Figure S2 minimize the off-target effects in CRISPR-Cas-mediated genome editing.
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Supplementary Figure S4 20. Komor AC, Kim YB, Packer MS, et al. Programmable editing of a target
Supplementary Table S1 base in genomic DNA without double-stranded DNA cleavage. Nature
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