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. 2022 May 9;13(1):2351.
doi: 10.1038/s41467-022-29989-9.

Targeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies

Zsolt Bodai  1 , Alena L Bishop  2 , Valentino M Gantz  2 , Alexis C Komor  3
Affiliations

Targeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies

Zsolt Bodai et al. Nat Commun. .

Abstract

Programmable double-strand DNA breaks (DSBs) can be harnessed for precision genome editing through manipulation of the homology-directed repair (HDR) pathway. However, end-joining repair pathways often outcompete HDR and introduce insertions and deletions of bases (indels) at the DSB site, decreasing precision outcomes. It has been shown that indel sequences for a given DSB site are reproducible and can even be predicted. Here, we report a general strategy (the "double tap" method) to improve HDR-mediated precision genome editing efficiencies that takes advantage of the reproducible nature of indel sequences. The method simply involves the use of multiple gRNAs: a primary gRNA that targets the wild-type genomic sequence, and one or more secondary gRNAs that target the most common indel sequence(s), which in effect provides a "second chance" at HDR-mediated editing. This proof-of-principle study presents the double tap method as a simple yet effective option for enhancing precision editing in mammalian cells.

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Conflict of interest statement

A.C.K. is a member of the SAB of Pairwise Plants, is an equity holder for Pairwise Plants and Beam Therapeutics, and receives royalties from Pairwise Plants, Beam Therapeutics, and Editas Medicine via patents licensed from Harvard University. A.C.K.’s interests have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. V.M.G. is a founder of and has equity interests in Synbal, Inc. and Agragene, Inc., companies that may potentially benefit from the research results described in this manuscript. V.M.G. also serves on both the company’s Scientific Advisory Board and the Board of Directors of Synbal, Inc. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. All authors are listed as inventors in a Regents of the University of California pending patent application (US63/243,260) on the use of secondary gRNAs to improve precision genome editing.

Figures

Fig. 1
Fig. 1. Schematic and initial results of the double tap method.
a Schematic overview of the double tap method. Cas9 introduces a DSB at a locus of interest using the primary guide RNA. HDR processes a subset of the DSBs into the desired outcome using a donor template (blue sequence). Concurrently, indels are introduced at the DSB site via end-joining pathways (red sequences). These undesired indel sequences are subsequently targeted with secondary gRNAs to improve overall yields of the desired outcome through a second DSB introduction and sequential HDR repair. b Indel sequences and their corresponding introduction efficiencies at the MMACHC site after transfecting HEK293T cells with Cas9 and a non-targeting gRNA (top), the primary gRNA plus a non-targeting gRNA (middle), or the primary gRNA plus a secondary gRNA targeted to the indel sequence indicated with the black arrow (bottom). c HDR-mediated genome editing efficiencies at the FANCF (in which a low-frequency indel was targeted), APOB1, and MMACHC sites when HEK293T cells are transfected with an ssODN and plasmids encoding Cas9, the primary gRNA, and either a non-targeting gRNA (NT, left) or secondary gRNA(s) (DT for “double tap”, right; two secondary gRNAs were used with the FANCF primary gRNA, and one secondary gRNA was used at the other two sites). Plotted are the percent of total DNA sequencing reads with the desired modification introduced (perfect HDR products without indels). d HDR-mediated genome editing efficiencies at the RNF2 locus when HEK293T cells are transfected with an ssODN and plasmids encoding Cas9, the primary gRNA, and either a non-targeting gRNA (NT, far left) or one (1 x DT), two (2 x DT), or three (3 x DT) secondary gRNAs. Values on whisker plots represent the lowest observation, lower quartile, median, upper quartile, and the highest observation of three independent replicates. Data were analyzed with univariate statistics (one-way ANOVA [one-sided]), and p values are labeled on the graphs.
Fig. 2
Fig. 2. Improvements in HDR-mediated genome editing with ssODNs using the double tap method.
a Shown are the percent of DNA sequencing reads with the desired modification introduced (perfect HDR products without indels) for cells treated with primary gRNA and a non-targeting gRNA (NT, left), or primary gRNA and secondary gRNA(s) (DT, right; three secondary gRNAs were used at the HIRA and RNF2 sites, two secondary gRNAs were used at the HEK2, HEK3 and FANCF sites, and one secondary gRNA was used at the APOB1, APOB2, PSMB, PCSK, SEC61B and MMACHC sites). Values on the whisker plots represent the lowest observation, lower quartile, median, upper quartile and the highest observation of three independent replicates. Data were analyzed with univariate statistics (one-way ANOVA [one-sided]) and p values are labeled on the graphs. b Average fold-change values plotted against the average of the total initial rates of the indels targeted by secondary gRNAs for all of the genomic loci tested in Figs. 1 and 2. Error bars represent the propagation of uncertainty of the SD for n = 3 biological replicates. c Shown are the relative changes in HDR (green) and NHEJ (blue) frequencies relative to the primary and non-targeting gRNA samples. Values and error bars represent the mean and propagation of uncertainty of the SD for n = 3 biological replicates. d Shown are total indel rates of all samples, with the specific indels targeted by secondary gRNAs shown in yellow, orange, and red (depending on how many secondary gRNAs were used for a particular site, there may only be yellow or yellow and orange bars). Blue represents indels not targeted by secondary gRNAs. Values and error bars represent the mean of the number of sequencing reads with indel sequences divided by the total number of sequencing reads ± SD for n = 3 biological replicates. In (c), (d), when the ssODN encoded a blocking mutation, the site is labeled with an “_B”.
Fig. 3
Fig. 3. Further characterization of the double tap method.
a Additive effects of double tap and previously developed HDR-improving methods were investigated at the MMACHC site. Shown are the percent of DNA sequencing reads with the desired modification introduced (perfect HDR products without indels) for cells treated with primary gRNA and a non-targeting gRNA (NT, left), or primary gRNA and secondary gRNA (DT, right; only one secondary gRNA was used at the MMACHC site). NT and DT samples were additionally treated with the small molecule HDR enhancer (Alt-R) or with a Cas9-CtIP fusion construct (Cas9-HE). DMSO-treated and no additive samples served as a base line for comparison (the Alt-R molecule is dissolved in a DMSO solution). b Shown are total indel rates of samples from (a), with the specific indels targeted by the secondary gRNA shown in yellow. Blue represents indels not targeted by a secondary gRNA. Values and error bars represent the mean of the number of sequencing reads with indel sequences divided by the total number of sequencing reads ± SD for n = 3 biological replicates. c Double tap improvements using Cas9:gRNA RNP complex at 3 sites. Shown are the percent of DNA sequencing reads with the desired modification introduced (perfect HDR products without indels) for cells treated with primary gRNA and a non-targeting gRNA (NT, left), or primary gRNA and secondary gRNA(s) (DT, right; three secondary gRNAs were used at the RNF2 site, two secondary gRNAs were used at the HEK3 site, and one secondary gRNA was used at the MMACHC site). d Analysis of zygosity of genome edited isogenic cells (n = 41 for each groups) at the MMACHC locus. Shown are the frequency of the indicated genome editing outcomes from each set of edited cells. Samples in (a–c) were analyzed by NGS after 72 h, and samples in (d) were clonally expanded and genotyped by NGS after 3 weeks. a, c Values on the whisker plots represent the lowest observation, lower quartile, median, upper quartile and the highest observation of three independent replicates. Data were analyzed with univariate statistics (one-way ANOVA [one-sided]) and p values are labeled on the graphs.
Fig. 4
Fig. 4. Improvements in gene knock-in with dsDNA donor templates using the double tap method.
Selected scatter plots of GFP fluorescence (y-axis) and cell forward scatter (x-axis), showing gating for GFP fluorescence for HEK293T cells transfected with plasmids encoding dsDNA donor template, Cas9, and non-targeting gRNA only (top), primary and non-targeting gRNAs (middle), or primary and secondary gRNAs (bottom) for the ACTB gene (a) and the LMNA gene (b). c Quantification of the percent of cells with GFP fluorescence in the GFP knock-in experiment for the ACTB (top) and LMNA (bottom) genes. NT stands for non-targeting, OG + NT stands for primary with non-targeting, and OG + DT stands for primary and gRNAs. One secondary gRNA was used at both sites. Values on the whisker plots represent the lowest observation, lower quartile, median, upper quartile and the highest observation of three independent replicates. Data were analyzed with univariate statistics (one-way ANOVA [one-sided]) and p values are labeled on the graphs.
Fig. 5
Fig. 5. Improvements in HDR-mediated genome editing with ssODNs using the double tap method in human erythroleukemic (K562) and human cervical cancer (HeLa) cell lines.
a HeLa or K562 cells were transfected with ssODN, Cas9-p2A-GFP plasmid, and gRNA plasmids. After 72 h, cells were enriched with FACS and analyzed by NGS and HDR-mediated genome editing efficiencies were quantified. Shown are the percent of DNA sequencing reads with the desired modification introduced (perfect HDR products without indels) for cells treated with primary gRNA and a non-targeting gRNA (NT, left), or primary gRNA and secondary gRNA(s) (DT, right; one secondary gRNA was used at both sites). Data from the MMACHC site are on the left and those from the APOB1 site are on the right. Data acquired from K562 cells are on the top and those from HeLa cells are on the bottom. Values on the whisker plots represent the lowest observation, lower quartile, median, upper quartile and the highest observation of three independent replicates. Data were analyzed with univariate statistics (one-way ANOVA [one-sided]) and p values are labeled on the graphs. b Shown are total indel rates of all samples, with the specific indels targeted by secondary gRNAs shown in yellow. Blue represents indels not targeted by secondary gRNAs. Values and error bars represent the mean of the number of sequencing reads with indel sequences divided by the total number of sequencing reads ± SD for n = 3 biological replicates. Data points are marked as circles when the ssODN encoded an extra blocking mutation, and as triangles when no additional mutation was installed.
Fig. 6
Fig. 6. Installation of disease relevant mutations in the HBB and HEXA genes using the double tap method.
a Shown are the percent of DNA sequencing reads with the desired modification introduced (perfect HDR products without indels) for cells treated with primary gRNA and a non-targeting gRNA (NT), or primary gRNA and secondary gRNA(s) (DT; three secondary gRNAs were used at the HBB5 site, and one secondary gRNA was used at the HBB1, HEXA2 and HEXA5 sites). b Shown are total indel rates of all samples from (a), with the specific indels targeted by secondary gRNAs shown in yellow, orange, and red (depending on how many secondary gRNAs were used for a particular site, there may only be yellow bars). Blue represents indels not targeted by secondary gRNAs. c HEK293T cells were transfected with plasmids encoding the prime editor and pegRNA only (PE2 sample), or pegRNA and nicking gRNA (PE3 sample) to introduce the same mutations as in (a). After 72 h, cells were analyzed by NGS to determine the efficiencies of introduction of the intended edit. Shown are the percent of DNA sequencing reads with the desired modification introduced (perfectly edited products without indels) for double tap samples from (a) (labeled as DT), PE2 treated cells (labeled as PE2), or PE3 treated cells (labeled as PE3). Values on the whisker plots in (a) and (c) represent the lowest observation, lower quartile, median, upper quartile and the highest observation of three independent replicates. Data were analyzed with univariate statistics (one-way ANOVA [one-sided]) and p values are labeled on the graphs. Values and error bars in (b) represent the mean of the number of sequencing reads with indel sequences divided by the total number of sequencing reads ± SD for n = 3 biological replicates. Data points are marked as circles when the ssODN encoded an extra blocking mutation, and as triangles when no additional mutation was installed. Data points are marked as squares for prime editing samples.
Fig. 7
Fig. 7. Assessment of off-target editing due to the double tap method.
a HEK293T cells were transfected with Cas9 and gRNA plasmids (non-targeting, primary, or secondary gRNAs). After 72 h, cells were analyzed by NGS at the primary (on-target) and all predicted off-target loci. Shown are total indel rates of all samples. The primary (on-target) loci are labeled as OG, while predicted off-target sites for primary gRNAs are labeled as OG_OT, and predicted off-target sites for secondary gRNAs are labeled as DT_OT on the y axis. The label on the x-axis indicates which gRNA the cells were transfected with; the secondary (DT), non-targeting (NT) or primary (OG). Only one gRNA was used at a time, b HEK293T cells were transfected with plasmids encoding Cas9-p2A-GFP, primary gRNA, and either non-targeting gRNA or secondary gRNA(s). As a control, HEK293T were transfected with plasmids encoding Cas9-P2A-GFP and non-targeting gRNA only. After 72 h cells were stained with propidium iodide to quantify cell viability FACS. The percentage of transfected cells (as determined by GFP fluorescence) that were viable are plotted with respect to the primary gRNA used (RNF2, HBB5, APOB1, and MMACHC). Samples with primary and non-targeting gRNAs are shown in blue, while those with primary and secondary gRNAs are in pink. Three secondary gRNAs were used with the RNF2 and HBB5 primary gRNA, and one secondary gRNA was used at APOB1 and MMACHC sites. The sample with non-targeting gRNA only is in green. Values and error bars represent the mean and standard deviation of viable cells within the transfected population for n = 3 biological replicates.
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