Peter Debye Institute for Soft Matter Physics, University of Leipzig, Leipzig 04103, Germany.
Institute of Microbiology & Archaea Centre, Single-Molecule Biochemistry Lab, University of Regensburg, Regensburg 93053, Germany.
Nucleic Acids Res. 2021 Dec 2;49(21):12411-12421. doi: 10.1093/nar/gkab1072.
CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 and its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different Streptococcus pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After initial target strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability than the pre-cleavage state. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around Cas9 bound to the target strand. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress.
CRISPR-Cas9 是一种核糖核蛋白复合物，能够特异性地结合并切割双链 DNA。由于其多功能性和可编程特异性，野生型 Cas9 及其切口酶和无切割活性的突变体已被用于各种生物学技术中。已经证明 Cas9 即使在单个 DNA 链被切割后，也能非常稳定地结合 DNA，从而抑制进一步的翻转并显著减缓体内修复过程。这在基因组编辑应用中构成了障碍。在这里，我们使用单分子磁镊来研究不同的酿脓链球菌 Cas9 变体在受到超螺旋挑战时的结合稳定性，这些变体在 DNA 被切割后。我们发现，不同的释放机制取决于被切割的 DNA 链。在初始靶链切割后，只有在 R 环崩塌后，超螺旋才会被移除。我们确定了 R 环具有不同稳定性的几个状态。最重要的是，我们发现 Cas9 的切割后状态表现出比切割前状态更高的稳定性。在非靶链切割后，非靶链围绕与靶链结合的 Cas9 旋转，超螺旋立即但缓慢释放。因此，即使在扭转应力升高的情况下，Cas9 及其非靶链切口酶突变体也能稳定地结合 DNA 数小时。
