Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity

Abstract

Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.

Figure 1. Effect of guide sequence extension on Cas9 activity

(A) Schematic showing Cas9 with matching or mismatching sgRNA sequences targeting the human EMX1 locus. (B) SURVEYOR assay gel showing comparable modification of target 1 by sgRNAs bearing 20- and 30-nt long guide sequences. (C) Northern blot showing that extended sgRNAs are largely reverted to 20-nt guide-length sgRNAs in HEK 293FT cells.

Figure 2. Double nicking facilitates efficient genome editing in human cells

(A) Schematic illustrating DNA double-stranded breaks using a pair of Cas9 D10A nickases (Cas9n). The D10A mutation renders Cas9 able to cleave only the strand complementary to the sgRNA; a pair of sgRNA-Cas9n complexes can nick both strands simultaneously. sgRNA offset is defined as the distance between the PAM-distal (5′) ends of the guide sequence of a given sgRNA pair. (B) Efficiency of double nicking induced NHEJ as a function of the offset distance between two sgRNAs. Sequences for all sgRNAs used can be found in Table S1. (n = 3; error bars show mean ± s.e.m.) (C) Representative sequences of the human EMX1 locus targeted by Cas9n. sgRNA target sites and PAMs are indicated by blue and magenta bars respectively. Below, selected sequences showing representative indels. See also Table S1 and S2.

Figure 3. Double nicking facilitates efficient genome editing in human cells

(A) Schematic illustrating DNA double strand breaks (red arrows) using Cas9 D10A nickases (Cas9n) and two sgRNAs. 5 off-target loci with sequence homology to EMX1 sgRNA 1 were selected to screen for Cas9n specificity. (B) On-target modification rate by Cas9n and a pair of sgRNAs is comparable to those mediated by wildtype Cas9 and single sgRNAs (left panel). Cas9-sgRNA1 complexes generate significant off-target mutagenesis, while no off-target locus modification is detected with Cas9n (right panel). (C) Levels of off-target modification with sgRNA 1 in HEK 293FT cells are measured by deep sequencing of five off-target loci. (D) Specificity comparison of Cas9n and wildtype Cas9 for sgRNA 1 off-target sites. Specificity ratio is calculated as on-target/off-target modification rates. (n = 3; error bars show mean ± s.e.m.) (E, F) Double nicking minimizes off-target modification at two human VEGFA loci while maintaining high specificity (on/off target modification ratio, n = 3; error bars show mean ± s.e.m.).

Figure 4. Double nicking allows insertion into the genome via HDR in human cells

(A) Schematic illustrating HDR mediated via a single stranded oligodeoxynucleotide (ssODN) template at a DSB created by a pair of Cas9n enzymes. Successful recombination at the DSB site introduces a HindIII restriction site. (B) Restriction digest assay gel showing successful insertion of HindIII cleavage sites by double nicking-mediated HDR in HEK 293FT cells. Upper bands are unmodified template; lower bands are HindIII cleavage product. (C) Double nicking promotes HDR in the HUES62 human embryonic stem cell line. HDR frequencies are determined by deep sequencing. (n = 3; error bars show mean ± s.e.m.). (D) HDR efficiency depends on the configuration of Cas9 or Cas9n-mediated nicks. HDR is facilitated when a nick occurs near the center of the ssODN homology arm leading to a 5′-resulting overhang. HDR-compatible nicking configurations are denoted by red arrows separated by overhang regions (black lines), and non-compatible configurations are shown with brown arrows and gray lines (top panel). HDR efficiency mediated by sgRNAs 22, 18, 21, 20, 15, 5 paired with either Cas9 or Cas9n is shown for comparison (bottom panel, Table S2).

Figure 5. Multiplexed nicking facilitates non-HR mediated gene integration and genomic deletions

(A) Schematic showing insertion of a double-stranded oligodeoxynucleotide (dsODN) donor fragment bearing overhangs complementary to 5′ overhangs created by Cas9 double nicking. The dsODN was designed to remove the native EMX1 stop codon and contains a HA tag, 3X FLAG tag, HindIII restriction site, Myc epitope tag, and a stop codon in frame, totaling 148 bp. Successful insertion was verified by Sanger sequencing as shown (1/37 clones screened). (B) Co-delivery of four sgRNAs with Cas9n generate long-range genomic deletions in the DYRK1A locus (from 0.5 kb up to 6 kb).

Figure 6. Cas9 double nicking mediates efficient indel formation in mouse embryos

(A) Schematic illustrating Cas9 double nicking targeting at the mouse Mecp2 locus. Representative indels are shown for mouse blastocysts co-injected with Cas9n-encoding mRNA and in vitro transcribed sgRNA pairs. (B) Efficient blastocyst modification is achieved at multiple concentrations of sgRNAs and wildtype Cas9 or Cas9n (titrating from 3 ng/uL to 100 ng/uL).