生物
移码突变
Cas9
清脆的
计算生物学
基因组编辑
遗传学
DNA测序
基因组
DNA
基因
突变
作者
Jonathan L. Schmid-Burgk,Linyi Gao,David Li,Zachary J G Gardner,Jonathan Strecker,Blake Lash,Feng Zhang
出处
期刊:Molecular Cell
[Elsevier]
日期:2020-05-01
卷期号:78 (4): 794-800.e8
被引量:137
标识
DOI:10.1016/j.molcel.2020.02.023
摘要
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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