Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases

Abstract

RNA-guided endonucleases (RGENs), derived from the prokaryotic adaptive immune system known as CRISPR/Cas, enable targeted genome engineering in cells and organisms. RGENs are ribonucleoproteins that consist of guide RNA and Cas9, a protein component originated from Streptococcus pyogenes. These enzymes cleave chromosomal DNA, whose sequence is complementary, to guide RNA in a targeted manner, producing site-specific DNA double-strand breaks (DSBs), the repair of which gives rise to targeted genome modifications. Despite broad interest in RGEN-mediated genome editing, these nucleases are limited by off-target mutations and unwanted chromosomal translocations associated with off-target DNA cleavages. Here, we show that off-target effects of RGENs can be reduced below the detection limits of deep sequencing by choosing unique target sequences in the genome and modifying both guide RNA and Cas9. We found that both the composition and structure of guide RNA can affect RGEN activities in cells to reduce off-target effects. RGENs efficiently discriminated on-target sites from off-target sites that differ by two bases. Furthermore, exome sequencing analysis showed that no off-target mutations were induced by two RGENs in four clonal populations of mutant cells. In addition, paired Cas9 nickases, composed of D10A Cas9 and guide RNA, which generate two single-strand breaks (SSBs) or nicks on different DNA strands, were highly specific in human cells, avoiding off-target mutations without sacrificing genome-editing efficiency. Interestingly, paired nickases induced chromosomal deletions in a targeted manner without causing unwanted translocations. Our results highlight the importance of choosing unique target sequences and optimizing guide RNA and Cas9 to avoid or reduce RGEN-induced off-target mutations.

Figure 1.

Mutation frequencies at on-target and potential off-target sites of the C4BPB- and CCR5-specific RGENs in K562 cells. (A,B) Cells were transfected with crRNA, tracrRNA, and the Cas9 plasmid or the Cas9 plasmid alone (negative control). PCR amplicons that span the on-target site and potential off-target sites were subjected to deep sequencing. Sequences that contained indels around the expected cleavage site were considered to be RGEN-induced mutations. Mismatched bases are shown in red. The PAM sequence is shown in blue. (C) In vitro cleavage assay of on-target or potential off-target sequences by the CCR5-specific RGEN. Plasmids that contain putative off-target (upper) or hybrid (middle) sequences were digested with the recombinant Cas9 protein complexed with crRNA and tracrRNA. Asterisks indicate cleaved DNA bands. (Bottom) DNA sequences of the on-target, off-target, and hybrid sites.

Figure 2.

Mutation frequencies at CCR5 on-target sites and CCR2 off-target sites. K562 cells were transfected with RGENs that target nine different sites. PCR amplicons that span the on-target and off-target sites were subjected to deep sequencing.

Figure 3.

Comparison of guide RNA structure. Mutation frequencies of the RGENs reported in Fu et al. (2013) were measured at on-target and off-target sites using the T7E1 assay. K562 cells were cotransfected with the Cas9-encoding plasmid and the plasmid encoding GX₁₉ sgRNA or GGX₂₀ sgRNA. Off-target sites (OT1-3, etc.) are labeled as in Fu et al. (2013).

Figure 4.

In vitro DNA cleavage by Cas9 nickases. (A) Schematic overview of the Cas9 nuclease and the paired Cas9 nickase. The PAM sequences and cleavage sites are shown in red. (B) Target sites in the human AAVS1 locus. The position of each target site is shown in red. (C) Schematic overview of DNA cleavage reactions. FAM dyes (shown in green) were linked to both 5′ ends of the DNA substrate. (D) DSBs and SSBs analyzed using fluorescent capillary electrophoresis. Fluorescently labeled DNA substrates were incubated with Cas9 nucleases or nickases before electrophoresis.

Figure 5.

Comparison of Cas9 nuclease and nickase behavior. (A) On-target mutation frequencies associated with Cas9 nucleases (WT), nickases (D10A), and paired nickases. Paired nickases that would produce 5′ overhangs or 3′ overhangs are indicated. (B) Analysis of off-target effects of Cas9 nucleases and paired nickases. A total of seven potential off-target sites for three sgRNAs were analyzed.

Figure 6.

DNA splicing induced by paired Cas9 nickases. (A) The target sites of paired nickases in the human AAVS1 locus. The distances between the AS2 site and each of the other sites are shown. Arrows indicate PCR primers. (B) Genomic deletions detected using PCR. Asterisks indicate deletion-specific PCR products. (C) DNA sequences of deletion-specific PCR products obtained using AS2 and L1 sgRNAs. Target site PAM sequences are shown in red, and sgRNA-matching sequences are shown in capital letters. Intact sgRNA-matching sequences are underlined. (D) A schematic model of paired Cas9 nickase-mediated chromosomal deletions. Newly synthesized DNA strands are shown in red.

Figure 7.

Paired Cas9 nickases do not induce translocations. (A) Schematic overview of chromosomal translocations between the on-target and off-target sites. (B) PCR amplification to detect chromosomal translocations. (C) Translocations induced by Cas9 nucleases but not by the nickase pair.