期刊
MOLECULAR CELL
卷 78, 期 4, 页码 794-+出版社
CELL PRESS
DOI: 10.1016/j.molcel.2020.02.023
关键词
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资金
- EMBO Long-Term Fellowship [ALTF 199-2017]
- Human Frontier Science Program
- NIH [1R01-HG009761, 1R01-MH110049, 1DP1-HL141201, 5R-M1HG006193-09]
- Howard Hughes Medical Institute
- Harold G. and Leila Mathers Foundation
- Poitras Center for Psychiatric Disorders Research at MIT
- Edward Mallinckrodt, Jr. Foundation
- Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT
- Phillips family
Determining the off-target cleavage profile of programmable nucleases is an important consideration for any genome editing experiment, and a number of Cas9 variants have been reported that improve specificity. We describe here tagmentation-based tag integration site sequencing (TTISS), an efficient, scalable method for analyzing double-strand breaks (DSBs) that we apply in parallel to eight Cas9 variants across 59 targets. Additionally, we generated thousands of other Cas9 variants and screened for variants with enhanced specificity and activity, identifying LZ3 Cas9, a high specificity variant with a unique +1 insertion profile. This comprehensive comparison reveals a general trade-off between Cas9 activity and specificity and provides information about the frequency of generation of +1 insertions, which has implications for correcting frameshift mutations.
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