Liu Shunhang, Rao Xichen, Zhao Ruiliang, Han Wenyuan
State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, China.
State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.
Front Genome Ed. 2022 Jul 26;4:929929. doi: 10.3389/fgeed.2022.929929. eCollection 2022.
Cas12a is a type V-A CRISPR-Cas RNA-guided endonuclease. It cleaves dsDNA at specific site, and then is activated for nonspecific ssDNA cleavage in trans . The immune function of the trans activity is still unknown. To address this question, we constructed a Cas12a targeting system in , where Cas12a cleaved a high-copy target plasmid to unleash the trans ssDNA cleavage activity. Then, we analyzed the effect of the Cas12a targeting on a non-target plasmid and a ssDNA phage. The results show that Cas12a efficiently eliminates target plasmid but exerts no impact on the maintenance of the non-target plasmid or plague formation efficiency of the phage. In addition, a two-spacer CRISPR array, which facilitates target plasmid depletion, still has no detectable effect on the non-target plasmid or phage either. Together, the data suggest that the trans ssDNA cleavage of Cas12a does not contribute to immunity .
Cas12a是一种V-A型CRISPR-Cas RNA引导的核酸内切酶。它在特定位点切割双链DNA，然后被激活以反式切割非特异性单链DNA。反式活性的免疫功能仍然未知。为了解决这个问题，我们构建了一个Cas12a靶向系统，在该系统中，Cas12a切割高拷贝的目标质粒以释放反式单链DNA切割活性。然后，我们分析了Cas12a靶向对非目标质粒和单链DNA噬菌体的影响。结果表明，Cas12a能有效消除目标质粒，但对非目标质粒的维持或噬菌体的噬菌斑形成效率没有影响。此外，促进目标质粒消耗的双间隔CRISPR阵列对非目标质粒或噬菌体也没有可检测到的影响。总之，数据表明Cas12a的反式单链DNA切割对免疫没有贡献。
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