The initiation, propagation and dynamics of CRISPR-SpyCas9 R-loop complex.
The initiation, propagation and dynamics of CRISPR-SpyCas9 R-loop complex.
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DOI：
10.1093/nar/gkx1117
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发表时间：
2018-01-09
影响因子：
14.9
通讯作者：
Lou J
中科院分区：
文献类型：
--
作者：
Zeng Y;Cui Y;Zhang Y;Zhang Y;Liang M;Chen H;Lan J;Song G;Lou J
CRISPR-Cas9系统已被广泛用于高效的基因组编辑。尽管Cas9蛋白与单引导RNA（sgRNA）和靶DNA复合的结构已经被解析，但关于Cas9内切核酸酶R环结构形成的分子细节仍然难以捉摸。在这里，我们使用各种靶序列检查化脓链球菌Cas9（SpyCas 9）及其突变体的DNA切割活性，并使用单分子荧光能量转移（smFRET）技术研究靶结合过程中R环结构的构象动力学。我们的结果显示，Cas9-sgRNA复合物将靶DNA分成几个不同的结构域：前间区序列邻近基序、接头、种子、中间和尾部。在种子配对后，Cas9瞬时保留半活性构象并诱导靶链或非靶链的切割。smFRET研究表明，在形成完全稳定的R-环复合物之前存在中间状态。动力学分析表明，这种新的中间状态的寿命随着向导DNA杂交双链体碱基配对长度的增加而增加，并在18 bp处达到最大值。这些数据为R环形成过程提供了新的见解，并揭示了CRISPR/Cas9系统中脱靶的来源。
CRISPR-Cas9 system has been widely used for efficient genome editing. Although the structures of Cas9 protein in complex with single-guided RNA (sgRNA) and target DNA have been resolved, the molecular details about the formation of Cas9 endonuclease R-loop structure remain elusive. Here we examine the DNA cleavage activities of Streptococcus pyogenes Cas9 (SpyCas9) and its mutants using various target sequences and study the conformational dynamics of R-loop structure during target binding using single-molecule fluorescence energy transfer (smFRET) technique. Our results show that Cas9–sgRNA complex divides the target DNA into several distinct domains: protospacer adjacent motif, linker, Seed, Middle and Tail. After seed pairing, the Cas9 transiently retains a semi-active conformation and induces the cleavage of either target or non-target strand. smFRET studies demonstrate that an intermediate state exists in prior to the formation of the fully stable R-loop complex. Kinetics analysis of this new intermediate state indicates that the lifetime of this state increases when the base-pairing length of guide-DNA hybrid duplex increases and reaches the maximum at the size of 18 bp. These data provide new insights into the process of R-loop formation and reveal the source of off-targeting in CRISPR/Cas9 system.
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