Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs

Abstract

CRISPR-Cas9 nucleases are powerful genome engineering tools, but unwanted cleavage at off-target and previously edited sites remains a major concern. Numerous strategies to reduce unwanted cleavage have been devised, but all are imperfect. Here, we report that off-target sites can be shielded from the active Cas9•single guide RNA (sgRNA) complex through the co-administration of dead-RNAs (dRNAs), truncated guide RNAs that direct Cas9 binding but not cleavage. dRNAs can effectively suppress a wide-range of off-targets with minimal optimization while preserving on-target editing, and they can be multiplexed to suppress several off-targets simultaneously. dRNAs can be combined with high-specificity Cas9 variants, which often do not eliminate all unwanted editing. Moreover, dRNAs can prevent cleavage of homology-directed repair (HDR)-corrected sites, facilitating scarless editing by eliminating the need for blocking mutations. Thus, we enable precise genome editing by establishing a flexible approach for suppressing unwanted editing of both off-targets and HDR-corrected sites.

Fig. 1. dRNA-mediated Off-Target Suppression (dOTS) effectively reduces off-target editing.

a Schematic representation of dOTS. A dRNA (green) with perfect complementarity for an off-target site directs Cas9 binding but not cleavage, protecting the site. b Indel frequencies and specificity ratios (on-target/off-target indel frequency ratios) at the FANCF-sgRNA2 on-target site and OT1 24 h after transfection of HEK-293T cells with Cas9, sgRNA, and FANCF-sgRNA2 OT1 dRNA1 or a nontargeting control dRNA (dNT) that does not target genomic DNA. For conditions without dRNA, an equivalent amount of pMAX-GFP was substituted. Means of n = 3 biological replicates depicted by solid lines. c Normalized specificity ratios, computed as the specificity ratio of the best dRNA condition (Supplementary Table 1) divided by the specificity ratio of the sgRNA only condition for 19 guide/off-target pairs tested in HEK-293T cells. Points depict the mean of n = 3 biological replicates, error bars show the standard error of the mean. d Indel frequencies and specificity ratios at the FANCF-sgRNA2 on-target site and OT1 24 h after transfection in U2OS cells, and e Elf1 embryonic stem cells. Control samples to the right of the x-axis break were performed separately. iCas9 denotes stable integration of Cas9 under the control of a doxycycline-inducible promoter. Means of n = 3 cell culture replicates depicted by solid lines. OT off-target. Source data are available in the Source Data file.

Fig. 2. dRNAs suppress off-target editing by competing with sgRNAs for off-target sites.

Representative gels of in vitro cleavage of PCR products containing either a FANCF-sgRNA2 OT1 or b the FANCF-sgRNA2 on-target site with either 150 or 450 fmoles of Cas9 FANCF-sgRNA2 RNP in the presence of variable amounts of the Cas9 FANCF-sgRNA2 OT1 dRNA1 complex. Fraction of uncut DNA determined by gel densitometry. Means of n = 3 replicates depicted by solid lines. For uncropped gels, see Supplementary Fig. 14. Source data are available in the Source Data file.

Fig. 3. dRNAs affect off-target, but not on-target, editing kinetics and can be titrated to improve specificity.

a Editing of FANCF-sgRNA2 on-target and OT1 sites using chemically inducible Cas9 (ciCas9) from 0 to 16 h after activation with A115. Nontargeting dRNA is a 14-base control dRNA targeting a non-endogenous site. NT = non-transfected control. Points depict the mean of n = 3 biological replicates. Error bars show the standard error of the mean. b Indel frequencies and specificity ratios at VEGFA sgRNA3 on-target and OT2 sites in cells transfected with plasmids encoding Cas9 and varying ratios of VEGFA sgRNA3 and dRNA2. dRNA untreated cells were transfected with Cas9 and a 1:1 VEGFA sgRNA3:GFP plasmid ratio. Error bars depict s.e.m. (n = 3 cell culture replicates). OT off-target. Source data are available in the Source Data file.

Fig. 4. dRNAs can be combined with other approaches for improving Cas9 specificity.

Indel frequencies and specificity ratios 24 h after transfection with a plasmids encoding WT Cas9, a dRNA targeting VEGFA sgRNA3 OT2 (dRNA2) and a truncated guide VEGFA tru-sgRNA3, or b High-specificity variants of Cas9 and a dRNA targeting FANCF-sgRNA2 OT1 (dRNA1). Wild-type Cas9 (WT), eSpCas9 (E), SpCas9-HF1 (HF1), HypaCas9 (Hypa). Means of n = 3 cell culture replicates depicted by solid lines. OT off-target. Source data are available in the Source Data file.

Fig. 5. dRNAs can be multiplexed to suppress several off-targets simultaneously.

Indel frequencies and specificity ratios 24 h after transfection of plasmids encoding either a wild-type (WT) or b eSpCas9 (E), VEGFA sgRNA2, and dRNAs targeting one of three VEGFA sgRNA2 off-targets (OT1 dRNA1, OT2 dRNA8, OT17 dRNA8). Means of n = 3 cell culture replicates depicted by solid lines. OT off-target. Source data are available in the Source Data file.

Fig. 6. dRNA on-target recutting suppression (dReCS) facilitates scarless HDR.

a Schematic depicting dReCS and alignment of BFP, GFP, sgRNA, and dRNA sequences. dRNA (green) exhibiting perfect complementarity for the repaired site directs Cas9 binding but not cleavage, protecting the repaired site. Single base change to generate GFP from BFP is displayed in green with affected codon indicated by gray box. PAM sequences are underlined. Black arrow indicates best dRNA, as determined by maximal improvement in HDR yield. b Indels and homology-directed repair (HDR) as assessed by flow cytometry, where indels lead to a loss of BFP signal, and HDR leads to a loss of BFP and gain of GFP signal. c HDR as a percentage of total Cas9 edits observed. Means of n = 3 cell culture replicates depicted by solid lines. dRNA BFP sgRNA1 dRNA3 (see Supplementary Fig. 13, Supplementary Data Set 1). Source data are available in the Source Data file.