A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets

Abstract

Streptococcus pyogenes Cas9 (SpCas9) has been employed as a genome engineering tool with a promising potential within therapeutics. However, its off-target effects present major safety concerns for applications requiring high specificity. Approaches developed to date to mitigate this effect, including any of the increased-fidelity (i.e., high-fidelity) SpCas9 variants, only provide efficient editing on a relatively small fraction of targets without detectable off-targets. Upon addressing this problem, we reveal a rather unexpected cleavability ranking of target sequences, and a cleavage rule that governs the on-target and off-target cleavage of increased-fidelity SpCas9 variants but not that of SpCas9-NG or xCas9. According to this rule, for each target, an optimal variant with matching fidelity must be identified for efficient cleavage without detectable off-target effects. Based on this insight, we develop here an extended set of variants, the CRISPRecise set, with increased fidelity spanning across a wide range, with differences in fidelity small enough to comprise an optimal variant for each target, regardless of its cleavability ranking. We demonstrate efficient editing with maximum specificity even on those targets that have not been possible in previous studies.

基因编辑设计和分析辅助工具的研究进展

“…另外, sgRNA的长度也会对Cas核酸酶的脱 靶率造成影响, 对于SpCas9, 常设计的靶点序列为20 nt, 而使用17 nt或者18 nt靶点序列的sgRNA, 能够显著减少 SpCas9进行基因编辑的脱靶率 [105] . 同时, Cas核酸酶的脱靶率与Cas蛋白有关, Klein-stiver等人 [106] 通过开发高保真的SpCas9, 即SpCas9-HF1, 可显著降低SpCas9的脱靶率; Kulcsár等人 [107] 对 SpCas9进行突变, 开发了一套SpCas9的变体, 即CRIS- . 研究表明, CBE系 统可在人类细胞中编辑时会产生严重的DNA和RNA脱 靶效应, 引起DNA和RNA链上的单核苷酸突变(single nucleotide variations, SNVs) [109,110] .…”

Recent Advances in Tomato Gene Editing

“…Furthermore, after Cas12a cleaves the target DNA, it produces a 5′ overhang, increasing the efficiency of precise gene editing in AT-rich target regions, which Cas9 has difficulty accessing [ 44 ]. Beyond that, while Cas9 and Cas12a exhibit similar mismatches in their target DNA when assessed in vitro [ 56 ], it has been observed that Cas12a displays a lower off-target effect than Cas9 when conducting gene editing [ 57 , 58 ]. Initial successful outcomes following gene editing in plants using Cpf1, which is the Cas12a ortholog from Francisella novicida , were achieved in 2016 for Oryza sativa and Nicotiana benthamiana [ 59 ].…”