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）具有多种裂解活性。在没有CRISPR RNA（crRNA）的情况下，我们研究了存在多种二价金属离子的Cas12a直系同源基因的DNase活性和底物特异性。来自不同物种的Cas12a能够降解单链DNA（ssDNA）和/或双链DNA（dsDNA），具体取决于所使用的金属离子。尽管共享多样Cas12a直向同源物，只是其中高序列相似性和功能性结构域氨基酸球菌属。Cas12a（AsCas12a）在切割ssDNA方面表现出主要优势，但在存在镁（II）离子的情况下，对dsDNA底物没有可检测的活性。此外，我们发现AsCas12a和Francisella novicida在锰（II）离子存在下，Cas12a（FnCas12a）引起了dsDNA的大量切割。更重要的是，DNase活性可以被具有硫代磷酸酯键修饰的合成DNA寡核苷酸抑制。总体而言，与crRNA指导的dsDNA断裂相比，Cas12a直系同源物的ssDNase活性揭示了一种独特的DNA切割方法，并为潜在的生物学和治疗应用提供了见识。