CRISPR-Cpf1对典型和非典型PAM识别的结构基础
Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1.
作者信息
Yamano Takashi, Zetsche Bernd, Ishitani Ryuichiro, Zhang Feng, Nishimasu Hiroshi, Nureki Osamu
机构信息
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
出版信息
Mol Cell. 2017 Aug 17;67(4):633-645.e3. doi: 10.1016/j.molcel.2017.06.035. Epub 2017 Aug 3.
The RNA-guided Cpf1 (also known as Cas12a) nuclease associates with a CRISPR RNA (crRNA) and cleaves the double-stranded DNA target complementary to the crRNA guide. The two Cpf1 orthologs from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) have been harnessed for eukaryotic genome editing. Cpf1 requires a specific nucleotide sequence, called a protospacer adjacent motif (PAM), for target recognition. Besides the canonical TTTV PAM, Cpf1 recognizes suboptimal C-containing PAMs. Here, we report four crystal structures of LbCpf1 in complex with the crRNA and its target DNA containing either TTTA, TCTA, TCCA, or CCCA as the PAM. These structures revealed that, depending on the PAM sequences, LbCpf1 undergoes conformational changes to form altered interactions with the PAM-containing DNA duplexes, thereby achieving the relaxed PAM recognition. Collectively, the present structures advance our mechanistic understanding of the PAM-dependent, crRNA-guided DNA cleavage by the Cpf1 family nucleases.
RNA引导的Cpf1（也称为Cas12a）核酸酶与CRISPR RNA（crRNA）结合，并切割与crRNA引导序列互补的双链DNA靶标。来自酸氨基球菌属（AsCpf1）和毛螺菌科细菌（LbCpf1）的两种Cpf1直系同源物已被用于真核基因组编辑。Cpf1需要一个特定的核苷酸序列，称为原间隔相邻基序（PAM），用于靶标识别。除了典型的TTTV PAM外，Cpf1还识别次优的含C的PAM。在这里，我们报告了LbCpf1与crRNA及其靶DNA形成复合物的四个晶体结构，其中靶DNA含有TTTA、TCTA、TCCA或CCCA作为PAM。这些结构表明，根据PAM序列，LbCpf1会发生构象变化，与含PAM的DNA双链体形成改变的相互作用，从而实现宽松的PAM识别。总体而言，目前的结构推进了我们对Cpf1家族核酸酶依赖PAM、crRNA引导的DNA切割机制的理解。
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