Department of Chemistry and Biochemistry, Price Family Foundation Institute of Structural Biology, University of Oklahoma, Stephenson Life Sciences Research Center, Norman, OK, USA.
Department of Chemistry, University of Southern California, Los Angeles, CA, USA.
FEBS Lett. 2021 Apr;595(7):892-912. doi: 10.1002/1873-3468.14051. Epub 2021 Feb 28.
Cas12a is an RNA-guided DNA endonuclease of the type V-A CRISPR-Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity in Streptococcus pyogenes (Spy) Cas9. Here, we examined a BH variant of Cas12a from Francisella novicida (FnoCas12a ) to test mechanistic conservation. Our results show that for RNA-guided DNA cleavage (cis-activity), FnoCas12a accumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12a also possess reduced trans-single-stranded DNA cleavage activity. These results implicate the BH in substrate selectivity in both cis- and trans-cleavages and show its conserved role in target discrimination among Cas nucleases.
Cas12a 是一种 RNA 指导的 DNA 内切酶，属于 V-A 型 CRISPR-Cas 系统，与 II 型 Cas9 蛋白具有趋同进化的特点。我们之前的研究表明，桥环螺旋（BH）中的脯氨酸取代赋予了酿脓链球菌 Cas9（Spy Cas9）对目标 DNA 的切割选择性。在这里，我们研究了来自弗朗西斯氏菌 novicida（FnoCas12a）的 Cas12a BH 变体，以测试其机制的保守性。我们的结果表明，对于 RNA 引导的 DNA 切割（顺式活性），FnoCas12a 在切割带有错配的超螺旋 DNA 底物时会积累缺口产物，某些错配位置对线性化更为不利。FnoCas12a 还具有降低的反式单链 DNA 切割活性。这些结果表明 BH 在顺式和反式切割的底物选择性中起作用，并显示其在 Cas 核酸酶的靶标识别中具有保守作用。
