用于研究 CRISPR Cas9 诱导的 DNA 解旋的单分子方法。

Single molecule methods for studying CRISPR Cas9-induced DNA unwinding.

机构信息

Department of Biology, Johns Hopkins University, Baltimore, MD, USA.

Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

出版信息

Methods. 2022 Aug;204:319-326. doi: 10.1016/j.ymeth.2021.11.003. Epub 2021 Nov 10.

DOI:10.1016/j.ymeth.2021.11.003

PMID:34767923

Abstract

Like helicases, CRISPR proteins such as Cas9 and Cas12a unwind DNA, but unlike helicases, these CRISPR proteins do not use ATP. Instead, they use binding energy to melt DNA locally and then utilize basepairing between guide (g) RNA and target strand to continue to unwind the DNA. CRISPR Cas9 is the most widely used tool for genome editing applications. The Cas9 endonuclease forms a complex with gRNA that can be programmed to bind a specific 20 bp segment of DNA, the protospacer. If there is enough of a sequence match between sgRNA and protospacer, Cas9 undergoes a conformational change, which activates the two nuclease domains, causing a double strand break in the DNA. We can use single-molecule FRET (smFRET) to probe the state of DNA unwinding as a function of mismatches between sgRNA and DNA. This approach can also be used to probe the position of Cas9's HNH domain before and after cleavage.

摘要

与解旋酶类似，CRISPR 蛋白（如 Cas9 和 Cas12a）也能解开 DNA，但与解旋酶不同的是，这些 CRISPR 蛋白不使用 ATP。相反，它们利用结合能局部融化 DNA，然后利用向导 (g) RNA 与靶链之间的碱基配对继续解开 DNA。CRISPR Cas9 是基因组编辑应用中最广泛使用的工具。Cas9 内切酶与 gRNA 形成复合物，可以被编程为结合特定的 20 bp 长度的 DNA，即原间隔序列。如果 sgRNA 和原间隔序列之间有足够的序列匹配，Cas9 会发生构象变化，激活两个核酸酶结构域，导致 DNA 双链断裂。我们可以使用单分子荧光共振能量转移 (smFRET) 来探测 sgRNA 与 DNA 之间的错配作为 DNA 解旋状态的函数。这种方法也可用于探测 Cas9 的 HNH 结构域在切割前后的位置。