CRISPR-Cas12a 系统对靶 DNA 和 crRNA 序列之间插入和缺失的脱靶效应,Analytical Chemistry

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CRISPR-Cas12a 系统对靶 DNA 和 crRNA 序列之间插入和缺失的脱靶效应
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-06-07 , DOI: 10.1021/acs.analchem.1c05499
Wei Zhang ₁ , Rui Shi ₁ , Kejun Dong ₁ , Hao Hu ₂ , Wan Shu ₁ , Yaoqin Mu ₂ , Bei Yan ₂ , Longjie Li _{2,

3} , Xianjin Xiao _{1,

2,

4,

5} , Hongbo Wang ₁

Affiliation

CRISPR-Cas12a系统是一种高效、靶向的新型基因组编辑工具。然而，基因组中的其他序列也可能被非特异性切割，导致不可避免的脱靶效应。因此，有必要进一步了解CRISPR-Cas12a识别靶序列的机制，以避免其脱靶效应。在这里，我们展示了目标 dsDNA 序列的插入（DNA 气泡）或缺失（RNA 气泡）与 crRNA 序列相比，CRISPR-Cas12a 系统仍然可以识别和切割目标 dsDNA 序列。我们得出结论，CRISPR-Cas12a对气泡的耐受性与气泡的位置和大小以及crRNA的GC碱基含量密切相关。此外，我们利用 CRISPR-Cas12a 的独特特性发明了一种检测突变的新方法，并成功检测到 CD41-42(-CTTT) 突变。该方法的检出限为0.001%。总体而言，我们的结果强烈表明，除了考虑由碱基错配引起的脱靶效应外，还需要对靶 dsDNA 序列的插入和缺失进行全面的脱靶分析，以及有效减少潜在脱靶切割的具体指导方针提出，完善CRISPR-Cas12a生物应用的安全手册。

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The Off-Target Effect of CRISPR-Cas12a System toward Insertions and Deletions between Target DNA and crRNA Sequences

The CRISPR-Cas12a system is a new type of genome editing tool with high efficiency and targeting. However, other sequences in the genome may also be cleaved nonspecifically, resulting in unavoidable off-target effects. Therefore, it is necessary to learn more about the mechanism of CRISPR-Cas12a to recognize target sequences to avoid its off-target effects. Here, we show that insertion (DNA bubble) or deletion (RNA bubble) of the target dsDNA sequence compared with the crRNA sequence, the CRISPR-Cas12a system can still recognize and cleave the target dsDNA sequence. We conclude that the tolerance of CRISPR-Cas12a to the bubbles is closely related to the location and size of the bubble and the GC base content of crRNA. In addition, we used the unique property of CRISPR-Cas12a to invent a new method to detect mutations and successfully detect the CD41–42(-CTTT) mutation. The detection limit of this method is 0.001%. Overall, our results strongly indicate that in addition to considering off-target effects caused by base mismatches, a comprehensive off-target analysis of the insertion and deletion of the target dsDNA sequence is required, and specific guidelines for effectively reducing potential off-target cleavage are proposed, to improve the safety manual of CRISPR-Cas12a biological application.

更新日期：2022-06-07

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