CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects
CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects
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DOI：
10.1074/jbc.ra120.012933
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发表时间：
2020-04-24
影响因子：
4.8
通讯作者：
Sashital, Dipali G.
中科院分区：
文献类型：
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作者：
Murugan, Karthik;Seetharam, Arun S.;Sashital, Dipali G.
Cas 12 a（Cpf 1）是细菌V-A型CRISPR-Cas抗噬菌体免疫系统中的RNA引导的核酸内切酶，其可以重新用于基因组编辑。Cas 12 a可以在体内以高特异性结合和切割dsDNA靶标，使其成为扩大用于精确基因组编辑的酶库的理想候选者。然而，这种报道的高特异性与Cas 12 a作为针对快速进化的免疫效应子的天然作用相矛盾。在这里，我们采用高通量体外切割测定来确定和比较三种不同的天然Cas 12 a直系同源物（FnCas 12 a、LbCas 12 a和AsCas 12 a）的天然切割特异性和活性。令人惊讶的是，我们观察到随机化靶文库的普遍序列特异性切口，其中在Cas 12 a靶向的DNA-RNA杂交序列中含有多达四个错配的DNA序列的强切口。我们还发现这些切口和切割活性取决于错配类型和位置，并随Cas 12 a直系同源物和CRISPR RNA序列而变化。我们的分析进一步揭示了当Cas 12 a通过与靶DNA结合而被激活时dsDNA的强非特异性切口。总之，我们的研究结果表明，Cas 12 a对dsDNA底物具有多种切口活性，并且这些活性在不同的Cas 12 a直系同源物中变化。
Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.