PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease.
PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease.
复制标题
C2C1 CRISPR-CAS核酸内切酶的PAM依赖性靶DNA识别和切割。
DOI：
10.1016/j.cell.2016.11.053
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发表时间：
2016-12-15
期刊：
影响因子：
64.5
通讯作者：
Patel DJ
中科院分区：
文献类型：
--
作者：
Yang H;Gao P;Rajashankar KR;Patel DJ
C2 c1是一种新发现的引导RNA介导的V-B型CRISPR-Cas内切核酸酶，其位点特异性靶向并切割靶DNA的两条链。我们已经确定了Alicyclobacillus acidoterrestris C2 c1（AacC 2c 1）的晶体结构结合到sgRNA作为一个二元复合物和目标DNA作为三元复合物，从而捕获催化活性构象的AacC 2c 1与目标和非目标DNA链独立定位在一个单一的RuvC催化口袋。此外，C2 c1介导的切割导致靶DNA的交错的七个核苷酸断裂。crRNA在二元复合物中采用预先排序的五核苷酸A型种子序列，释放插入的色氨酸，促进20-bp RNA-DNA异源双链体在三元复合物形成时的拉链。值得注意的是，PAM相互作用的裂缝采取了“锁定”的三元复合物形成的构象。C2 c1三元复合物与它们的Cas9和Cpf 1对应物的结构比较突出了这些不同的CRISPR-Cas9系统所采用的不同机制，从而扩大和增强了它们作为基因组编辑工具的适用性。对C2 c1 RNA引导的DNA内切酶的结构分析揭示了识别和切割靶DNA的独特模式，指出了与其他CRISPR效应子（如Cas9和Cpf 1）的机制差异，这可能对新的基因编辑应用产生影响。
C2c1 is a newly-identified guide RNA-mediated type V-B CRISPR-Cas endonuclease that site-specifically targets and cleaves both strands of target DNA. We have determined crystal structures of Alicyclobacillus acidoterrestris C2c1 (AacC2c1) bound to sgRNA as a binary complex and to target DNAs as ternary complexes, thereby capturing catalytically competent conformations of AacC2c1 with both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. Moreover, C2c1-mediated cleavage results in a staggered seven-nucleotide break of target DNA. crRNA adopts a pre-ordered five-nucleotide A-form seed sequence in the binary complex, with release of an inserted tryptophan, facilitating zippering-up of 20-bp RNA-DNA heteroduplex on ternary complex formation. Notably, the PAM-interacting cleft adopts a “locked” conformation on ternary complex formation. Structural comparison of C2c1 ternary complexes with their Cas9 and Cpf1 counterparts highlights the diverse mechanisms adopted by these distinct CRISPR-Cas9 systems, thereby broadening and enhancing their applicability as genome editing tools. Structural analyses of a C2c1 RNA-guided DNA endonuclease reveal a distinctive mode for recognizing and cleaving target DNA, pointing to mechanistic differences from other CRISPR effectors like Cas9 and Cpf1 that may have implications for new gene editing applications.