DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.
DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.
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CRISPR相关的酶Cas9是一种RNA引导的内切核酸酶，使用RNA：DNA碱基对靶向细菌中的异物DNA。 CAS9：引导RNA复合物也是动物和植物中有效的基因组工程剂。在这里，我们使用单分子和大量生化实验来确定CAS9：RNA如何询问DNA以找到特定的切割位点。我们表明，Cas9：RNA的DNA结合和裂解都需要识别短的三核苷酸原子能探针邻近基序（PAM）。 cas9：rna忽略了与引导RNA的非目标DNA结合亲和力尺度，序列完全互补，但缺乏附近的PAM。 DNA链的分离和RNA：DNA异形形成在PAM处启动，并向目标序列的远端方向前进。此外，PAM相互作用触发CAS9催化活性。这些结果揭示了CAS9在扫描大型DNA分子时如何采用PAM识别来快速识别潜在的靶位点，并调节DSDNA分裂。
The CRISPR-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA:DNA base-pairing to target foreign DNA in bacteria. Cas9:guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9:RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9:RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9:RNA. DNA strand separation and RNA:DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 employs PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate dsDNA scission.