Li Bin, Yan Jingyue, Zhang Youxi, Li Wenqing, Zeng Chunxi, Zhao Weiyu, Hou Xucheng, Zhang Chengxiang, Dong Yizhou
Division of Pharmaceutics and Pharmacology, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA; Department of Infectious Disease, Shenzhen People's Hospital, The Second Clinical Medical College of Jinan University, The First Affiliated Hospital of Southern University of Science and Technology, Shenzhen 518020, China.
Division of Pharmaceutics and Pharmacology, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
Mol Ther Nucleic Acids. 2020 Mar 6;19:1043-1052. doi: 10.1016/j.omtn.2019.12.038. Epub 2020 Jan 14.
CRISPR-Cas12a (CRISPR-Cpf1) was reported to have multiple types of cleavage activities. Without the assistance of CRISPR RNA (crRNA), we investigated DNase activity and substrate specificity of Cas12a orthologs in the presence of diverse divalent metal ions. Cas12a from different species are capable of degrading single-stranded DNA (ssDNA) and/or double-stranded DNA (dsDNA), depending on the metal ions used. In spite of sharing high sequence similarity and functional domains among diverse Cas12a orthologs, only Acidaminococcus sp. Cas12a (AsCas12a) showed a predominant preference for cleaving ssDNA, but no detectable activity toward dsDNA substrate in the presence of magnesium (II) ions. In addition, we found that both AsCas12a and Francisella novicida Cas12a (FnCas12a) caused substantial dsDNA cleavage in the presence of manganese (II) ion. More importantly, the DNase activities can be inhibited by synthetic DNA oligonucleotides with phosphorothioate linkage modifications. Overall, ssDNase activity of the Cas12a orthologs uncovered a distinct approach for DNA cleavage compared with crRNA-guided dsDNA breaks, and provided insights into potential biological and therapeutic applications.
据报道，CRISPR-Cas12a（CRISPR-Cpf1）具有多种切割活性。在没有CRISPR RNA（crRNA）协助的情况下，我们研究了不同二价金属离子存在时Cas12a直系同源物的脱氧核糖核酸酶活性和底物特异性。不同物种的Cas12a能够降解单链DNA（ssDNA）和/或双链DNA（dsDNA），这取决于所使用的金属离子。尽管不同的Cas12a直系同源物之间具有高度的序列相似性和功能结构域，但只有酸氨基球菌属Cas12a（AsCas12a）表现出对切割ssDNA的主要偏好，而在镁（II）离子存在下对dsDNA底物没有可检测到的活性。此外，我们发现AsCas12a和新凶手弗朗西斯菌Cas12a（FnCas12a）在锰（II）离子存在下都会导致大量的dsDNA切割。更重要的是，脱氧核糖核酸酶活性可以被具有硫代磷酸酯连接修饰的合成DNA寡核苷酸抑制。总体而言，Cas12a直系同源物的ssDNA酶活性揭示了一种与crRNA引导的dsDNA断裂相比截然不同的DNA切割方法，并为潜在的生物学和治疗应用提供了见解。
