Kiernan Kaitlyn A, Kwon Jieun, Merrill Bradley J, Simonović Miljan
Department of Biochemistry and Molecular Genetics, University of Illinois Chicago, 900 S Ashland Ave, Chicago, IL 60607, USA.
Nucleic Acids Res. 2025 Jan 7;53(1). doi: 10.1093/nar/gkae1164.
The efficiency and accuracy of CRISPR-Cas9 targeting varies considerably across genomic targets and remains a persistent issue for using this system in cells. Studies have shown that the use of 5' truncated single guide RNAs (sgRNAs) can reduce the rate of unwanted off-target recognition while still maintaining on-target specificity. However, it is not well-understood how reducing target complementarity enhances specificity or how truncation past 15 nucleotides (nts) prevents full Cas9 activation without compromising on-target binding. Here, we use biochemistry and cryogenic electron microscopy to investigate Cas9 structure and activity when bound to a 14-nt sgRNA. Our structures reveal that the shortened path of the displaced non-target strand (NTS) sterically occludes docking of the HNH L1 linker and prevents proper positioning of the nuclease domains. We show that cleavage inhibition can be alleviated by either artificially melting the protospacer adjacent motif (PAM)-distal duplex or providing a supercoiled substrate. Even though Cas9 forms a stable complex with its target, we find that plasmid cleavage is ∼1000-fold slower with a 14-nt sgRNA than with a full-length 20-nt sgRNA. Our results provide a structural basis for Cas9 target binding with 5' truncated sgRNAs and underline the importance of PAM-distal NTS availability in promoting Cas9 activation.
CRISPR-Cas9靶向的效率和准确性在不同基因组靶点之间差异很大，并且在细胞中使用该系统时仍然是一个长期存在的问题。研究表明，使用5'端截短的单向导RNA（sgRNA）可以降低意外脱靶识别的发生率，同时仍能保持靶向特异性。然而，目前尚不清楚减少靶点互补性如何提高特异性，也不清楚超过15个核苷酸（nt）的截短如何在不影响靶向结合的情况下阻止Cas9的完全激活。在这里，我们利用生物化学和低温电子显微镜技术研究了与14-nt sgRNA结合时Cas9的结构和活性。我们的结构显示，被置换的非靶向链（NTS）缩短的路径在空间上阻碍了HNH L1连接子的对接，并阻止了核酸酶结构域的正确定位。我们表明，通过人工解链原间隔序列相邻基序（PAM）远端双链体或提供超螺旋底物，可以缓解切割抑制。尽管Cas9与其靶点形成了稳定的复合物，但我们发现，与全长20-nt sgRNA相比，14-nt sgRNA的质粒切割速度慢约1000倍。我们的结果为Cas9与5'端截短的sgRNA的靶点结合提供了结构基础，并强调了PAM远端NTS可用性在促进Cas9激活中的重要性。
