Here is a literature review of CRISPR off-target (OT) mutation analysis in mouse oocytes. This review only concerns published experiments using “native” Cas9 that cuts both DNA strands, and not nickase-Cas9 experiments. Although nickase-Cas9 is much less prone to OT mutation, editing is still generally less efficient than with native Cas9 . Therefore it’s important to know whether the potential problem of off-target mutation rates with native Cas9 outweigh its utility. All the data below is pertinent to mouse zygotes. Other systems such as cell lines may have different OT rates.
Here’s a few pertinent questions to preface this review: First, how should potential off-targets (OTs) be defined ahead of time? It’s complicated by the fact that mismatches are less tolerated within the “seed” region of 8-12 bases proximal to the PAM site, and more tolerated in the more distal (5’) region of the protospacer. So some groups define OTs as having perfect matches to the seed region, while other groups defined them as simply having fewer than a threshold maximum number of mismatches anywhere in the protospacer. Alternatively, they can be scored for cleavage potential by algorithms such as the MIT CRISPR design tool.
Second, how is CRISPR performed? Some of these groups used RNA or DNA injections; most used slightly varying injection concentrations.
Third, how were the OTs screened? Most of these did direct sequencing on PCR from founders or Surveyor-type assays. Also, OTs often cut at lower efficiency than the on-target but the results depend on the assay sensitivity and the number of pups screened, which varies across studies. So the data here is only a general comparison.
I’m not focusing on those differences here, since the overall picture is broadly similar - OT rates were generally low to nil.
Let’s start with the pair of 2013 Cell papers from the Jaenisch lab.
1. Wang et al. (Cell 2013) was the first report of CRISPR-mediated mutagenesis in mouse zygotes. They only considered OTs with perfect matches to the 12 bases adjacent to the PAM and also the PAM itself (NGG). For 2 targets, they defined 7 total OTs. (A third gRNA they used had no OTs by this definition). In 7 mutants pups carrying mutations at 2 simultaneously-targeted gRNA targets, they found zero mutations at the 7 OTs.
Bottom line: 7 OTs screened, 0 mutated.
2. Yang et al (Cell 2013) screened OTs that were defined at having “up to 3 or 4” mismatches. (In my experience most mouse CRISPR targets have several-to-many 3-base mismatches, and I’m guessing that most targets will have many 4-base mismatches in mammalian genomes.) I believe they screened OTs for 5 targets across 4 different genes. A total of 35 pups and ES cell clones were screened in separate experiments using different gRNAs. Of 47 OTs, only 3 had mutations. Two of those sites were mutated in multiple mice, indicating fairly high rates at these particular OTs. However, the mutated OTs all had only 2 or 1 mismatches, and the “high rate” OTs only had 1 mismatch near the 5’ end, distal to the seed region.
Bottom line: 47 OTs screened, 3 mutated.
3. Li et al. (Nat. Biotech. 2013). Of 4 targets they used, only two had OTs with fewer than 4 mismatches or perfect seed matches, so they focused on those. 12 founders were screened. In a subset of pups also screened some more OTs that had perfect seed matches but were otherwise totally mismatched.
Bottom line: more than 13 OTs screened, 0 mutated.
4. Mashiko et al (Sci. Rep. 2013) were the first to publish on injecting plasmid DNAs into mouse zygotes for transient CRISPR expression. Similar to Wang et al, they defined OTs as having a perfect match to the 12-13 bases adjacent to the PAM. For two targets, they defined 7 and 4 OTs respectively; in 16 and 8 mutant pups made with either gRNA, they found one pup with a single OT mutation.
Bottom line: 11 OTs screened, 1 mutated.
5. Fujii et al (NAR 2013) targeted the Rosa26 locus, and reported that OT rates dropped as injected RNA concentrations were lowered. They inspected 10 OTs with “3 or 4 mismatches” but each of these actually had a mismatch to the “N” of the PAM motif, which doesn’t affect targeting, so these were really “2” and “3” mismatched OTs. No OT mutations were found for the true 3-mismatch OTs. However they found mutations in all four 2-mismatch OTs, particularly when injecting higher RNA concentrations . They also examined 12 OTs for 2 targets in Hprt and found no OT mutations.
Bottom line: 22 OTs screened; 4 mutated but only in “2-mismatch” OTs.
6. Wu et al (Cell Stem Cell 2013) targeted the Crygc gene and defined OTs as having no mismatches in a 14 base seed region of the target. Of 10 OTs, 1 was mutated in 2 out of 12 pups.
Bottom line: 10 OTs screened, 1 mutated.
7. Inui et al (Sci. Rep. 2014) examined about 10 OTs total for two targets, in Sox9 and Sf-1.
Bottom line: ~10 OTs screened, 0 mutated.
8. Zhou et al (FEBS J. 2014) retargeted an EGFP cassette separately with two gRNAs. OTs were defined using the MIT CRISPR design tool, and they analyzed a subset of these (15 OTs per target) by surveyor assay on the founder pups. For one target, they detected mutations in 4 OTs, but not in any OTs for the second target.
Bottom line: 30 OTs screened, 4 mutated.
9. Mizuno et al (Mamm. Genome 2014) targeted the Tyr gene and screened 5 OTs in founders by sequencing.
Bottom line: 5 OTs screened, 0 mutated.
10. Han et al (RNA Biol. 2014) used 4 targets and identified OTs with the MIT CRISPR design tool. They screened 3 founders for the “top 5” potential OTs.
Bottom line: 20 OTs screened, 0 mutated.
Here’s a quasi-meta-analysis:
From these 10 studies, 5 (50%) were able to detect some degree of off-target mutation.
But from ~175 OT’s screened, mutations in only 13 (7%) were detected. Several of these OTs had fewer than 3 mismatches to the target.
In conclusion, the consensus from many studies of CRISPR-mediated mouse engineering demonstrates that native Cas9 has a low rate of off-target effects in mouse zygotes. Of course, targets should still be pre-screened when possible to avoid those that will have more potential off-targets, particularly those with fewer than 3 mismatches within the protospacer.
Doug Mortlock 2014.
1. Wang, H., H. Yang, C.S. Shivalila, M.M. Dawlaty, A.W. Cheng, F. Zhang, and R. Jaenisch, One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell, 2013. 153(4): p. 910-8.
2. Yang, H., H. Wang, C.S. Shivalila, A.W. Cheng, L. Shi, and R. Jaenisch, One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell, 2013. 154(6): p. 1370-9.
3. Li, D., Z. Qiu, Y. Shao, Y. Chen, Y. Guan, M. Liu, Y. Li, N. Gao, L. Wang, X. Lu, and Y. Zhao, Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol, 2013. 31(8): p. 681-3.
4. Mashiko, D., Y. Fujihara, Y. Satouh, H. Miyata, A. Isotani, and M. Ikawa, Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Sci Rep, 2013. 3: p. 3355.
5. Fujii, W., K. Kawasaki, K. Sugiura, and K. Naito, Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease. Nucleic Acids Res, 2013. 41(20): p. e187.
6. Wu, Y., D. Liang, Y. Wang, M. Bai, W. Tang, S. Bao, Z. Yan, D. Li, and J. Li, Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell, 2013. 13(6): p. 659-62.
7. Inui, M., M. Miyado, M. Igarashi, M. Tamano, A. Kubo, S. Yamashita, H. Asahara, M. Fukami, and S. Takada, Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system. Sci Rep, 2014. 4: p. 5396.
8. Zhou, J., J. Wang, B. Shen, L. Chen, Y. Su, J. Yang, W. Zhang, X. Tian, and X. Huang, Dual sgRNAs facilitate CRISPR/Cas9-mediated mouse genome targeting. FEBS J, 2014. 281(7): p. 1717-25.
9. Mizuno, S., T.T. Dinh, K. Kato, S. Mizuno-Iijima, Y. Tanimoto, Y. Daitoku, Y. Hoshino, M. Ikawa, S. Takahashi, F. Sugiyama, and K. Yagami, Simple generation of albino C57BL/6J mice with G291T mutation in the tyrosinase gene by the CRISPR/Cas9 system. Mamm Genome, 2014. 25(7-8): p. 327-34.
10. Han, J., J. Zhang, L. Chen, B. Shen, J. Zhou, B. Hu, Y. Du, P.H. Tate, X. Huang, and W. Zhang, Efficient in vivo deletion of a large imprinted lncRNA by CRISPR/Cas9. RNA Biol, 2014. 11(7): p. 829-35.