Site-Specific Labeling Reveals Cas9 Induces Partial Unwinding Without RNA/DNA Pairing in Sequences Distal to the PAM
Site-Specific Labeling Reveals Cas9 Induces Partial Unwinding Without RNA/DNA Pairing in Sequences Distal to the PAM
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DOI：
10.1089/crispr.2021.0100
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发表时间：
2022-03-23
期刊：
影响因子：
3.7
通讯作者：
Qin, Peter Z.
中科院分区：
文献类型：
--
作者：
Li, Yue;Liu, Yukang;Qin, Peter Z.
CRISPR-Cas9是一种RNA引导的核酸酶，已广泛适用于基因组工程。Cas9靶选择中的关键决定因素是DNA双链体解旋以形成R环，其中单链RNA引导物与DNA链之一杂交。为了进一步理解Cas9对DNA解旋的作用，我们结合了两种类型的光谱标记，2-氨基嘌呤和氮氧自由基自旋标记，以研究由化脓链球菌Cas9诱导的特定DNA碱基对的解旋。从13到20个核苷酸的RNA指导长度变化所获得的数据显示，原型间隔区相邻基序远端的DNA片段可以采用“部分解旋”状态，其中DNA配对和DNA解旋群体的混合物平衡存在。显著的解旋可以发生在不受RNA/DNA配对支持的位置，并且解旋的程度取决于RNA指导长度并调节DNA切割活性。这些结果揭示了Cas9靶点的选择，并可能为基因组工程策略的发展提供信息。
CRISPR-Cas9 is an RNA-guided nuclease that has been widely adapted for genome engineering. A key determinant in Cas9 target selection is DNA duplex unwinding to form an R-loop, in which the single-stranded RNA guide hybridizes with one of the DNA strands. To advance understanding on DNA unwinding by Cas9, we combined two types of spectroscopic label, 2-aminopurine and nitroxide spin-label, to investigate unwinding at a specific DNA base pair induced by Streptococcus pyogenes Cas9. Data obtained with RNA guide lengths varying from 13 to 20 nucleotide revealed that the DNA segment distal to the protospacer adjacent motif can adopt a "partial unwinding" state, in which a mixture of DNA-paired and DNA-unwound populations exist in equilibrium. Significant unwinding can occur at positions not supported by RNA/DNA pairing, and the degree of unwinding depends on RNA guide length and modulates DNA cleavage activity. The results shed light on Cas9 target selection and may inform developments of genome-engineering strategies.