Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas12a is widely used for genome editing and diagnostics, so it is important to understand how RNA-guided DNA recognition activates the cleavage of the target strand (TS) following non-target-strand (NTS) cleavage. Here we used single-molecule magnetic tweezers, gel-based assays and nanopore sequencing to explore DNA unwinding and cleavage. In addition to dynamic and heterogenous R-loop formation, we also directly observed transient double-stranded DNA unwinding downstream of the 20-bp heteroduplex and, following NTS cleavage, formation of a hyperstable ‘clamped’ Cas12a–DNA intermediate necessary for TS cleavage. Annealing of a 4-nucleotide 3′ CRISPR RNA overhang to the unwound TS downstream of the heteroduplex inhibited clamping and slowed TS cleavage by ~16-fold. Alanine substitution of a conserved aromatic amino acid in the REC2 subdomain that normally caps the R-loop relieved this inhibition but favoured stabilisation of unwound states, suggesting that the REC2 subdomain regulates access of the 3′ CRISPR RNA to downstream DNA.

成簇规则间隔短回文重复序列 (CRISPR) – Cas12a 广泛用于基因组编辑和诊断，因此了解 RNA 引导的 DNA 识别如何激活非目标链 (NTS) 后目标链 (TS) 的切割非常重要) 裂解。在这里，我们使用单分子磁力镊子、基于凝胶的测定和纳米孔测序来探索 DNA 解旋和切割。除了动态和异质的 R 环形成之外，我们还直接观察到 20 bp 异源双链下游的瞬时双链 DNA 解旋，并在 NTS 切割后形成 TS 切割所需的超稳定“钳位”Cas12a-DNA 中间体。将 4 核苷酸 3' CRISPR RNA 突出端退火到异源双链体下游未缠绕的 TS 上，可抑制夹紧并使 TS 裂解速度减慢约 16 倍。通常覆盖 R 环的 REC2 子结构域中保守芳香族氨基酸的丙氨酸取代缓解了这种抑制，但有利于解开状态的稳定，表明 REC2 子结构域调节 3' CRISPR RNA 与下游 DNA 的接触。