A gate and clamp regulate sequential DNA strand cleavage by CRISPR-Cas12a
A gate and clamp regulate sequential DNA strand cleavage by CRISPR-Cas12a
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DOI：
10.1101/2021.06.18.448962
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发表时间：
2021-06
期刊：
影响因子：
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通讯作者：
Mohsin M. Naqvi;Laura J Lee;O. E. Torres Montaguth;M. Szczelkun
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作者：
Mohsin M. Naqvi;Laura J Lee;O. E. Torres Montaguth;M. Szczelkun
CRISPR-Cas 12 a已广泛用于基因组编辑和诊断应用，但尚未完全理解RNA引导的DNA识别如何激活非靶链（NTS）的顺序切割，然后是靶链（TS）。在这里，我们使用了单分子磁镊显微镜，系综凝胶为基础的分析和纳米孔测序，以探索DNA解旋和切割的耦合。除了动态R-环形成之外，我们还直接观察到20 bp DNA：RNA杂合体下游的瞬时dsDNA解旋，并且在NTS切割之后和TS切割之前，形成耐DNA扭曲的超稳定的“夹紧的”Cas 12 a-DNA中间体。丙氨酸取代的保守的芳香族氨基酸“门”在REC 2域，通常帽的异源双链产生更频繁和延长下游DNA呼吸，一个更长的寿命扭转抗性状态，和16倍的速度更快的TS切割。我们认为，呼吸和夹紧事件，调节门和NTS切割，提供解绕TS的RuvC核酸酶和结果从先前描述的REC 2和NUC域运动。
CRISPR-Cas12a has been widely used for genome editing and diagnostic applications, yet it is not fully understood how RNA-guided DNA recognition activates the sequential cleavage of the non-target strand (NTS) followed by the target strand (TS). Here we used single-molecule magnetic tweezers microscopy, ensemble gel-based assays and nanopore sequencing to explore the coupling of DNA unwinding and cleavage. In addition to dynamic R-loop formation, we also directly observed transient dsDNA unwinding downstream of the 20 bp DNA:RNA hybrid and, following NTS cleavage and prior to TS cleavage, formation of a hyperstable “clamped” Cas12a-DNA intermediate resistant to DNA twisting. Alanine substitution of a conserved aromatic amino acid “gate” in the REC2 domain that normally caps the heteroduplex produced more frequent and extended downstream DNA breathing, a longer-lived twist-resistant state, and a 16-fold faster rate of TS cleavage. We suggest that both breathing and clamping events, regulated by the gate and by NTS cleavage, deliver the unwound TS to the RuvC nuclease and result from previously described REC2 and NUC domain motions.