Department of Chemistry, University of Southern California, Los Angeles, California 90089, United States.
Department of Quantitative and Computational Biology, University of Southern California, Los Angeles, California 90089, United States.
Biochemistry. 2023 Sep 5;62(17):2541-2548. doi: 10.1021/acs.biochem.3c00250. Epub 2023 Aug 8.
CRISPR-Cas9 has been adapted as a readily programmable genome manipulation agent, and continuing technological advances rely on an in-depth mechanistic understanding of Cas9 target discrimination. Cas9 interrogates a target by unwinding the DNA duplex to form an R-loop, where the RNA guide hybridizes with one of the DNA strands. It has been shown that RNA guides shorter than the normal length of 20-nucleotide (-nt) support Cas9 cleavage activity by enabling partial unwinding beyond the RNA/DNA hybrid. To investigate whether DNA segment beyond the RNA/DNA hybrid can impact Cas9 target discrimination with truncated guides, Cas9 double-stranded DNA cleavage rates () were measured with 16-nt guides on targets with varying sequences at +17 to +20 positions distal to the protospacer-adjacent-motif (PAM). The data reveal a log-linear inverse correlation between and the PAM+(17-20) DNA duplex dissociation free energy (Δ), with sequences having smaller Δ showing faster cleavage and a higher degree of unwinding. The results indicate that, with a 16-nt guide, "peripheral" DNA sequences beyond the RNA/DNA hybrid contribute to target discrimination by tuning the cleavage reaction transition state through the modulation of PAM-distal unwinding. The finding provides mechanistic insights for the further development of strategies that use RNA guide truncation to enhance Cas9 specificity.
CRISPR-Cas9 已被改编为一种易于编程的基因组操作工具，而持续的技术进步依赖于对 Cas9 靶标识别的深入机制理解。Cas9 通过解开 DNA 双链形成 R 环来探测靶标，其中 RNA 向导与 DNA 链之一杂交。已经表明，短于正常 20 核苷酸（-nt）长度的 RNA 向导通过允许 RNA/DNA 杂交体之外的部分解旋来支持 Cas9 切割活性。为了研究 RNA/DNA 杂交体之外的 DNA 片段是否可以用截断的向导影响 Cas9 靶标识别，在距离原间隔基序相邻基序（PAM）+17 到+20 位的靶标上用 16-nt 向导测量 Cas9 双链 DNA 切割速率（）。数据显示与 PAM+（17-20）DNA 双链离解自由能（Δ）之间存在对数线性反比关系，其中具有较小Δ的序列显示出更快的切割和更高程度的解旋。结果表明，对于 16-nt 向导，RNA/DNA 杂交体之外的“外围”DNA 序列通过调节 PAM 远端解旋来调节切割反应过渡态，从而有助于靶标识别。这一发现为使用 RNA 向导截断来增强 Cas9 特异性的策略的进一步发展提供了机制见解。
