工程化CRISPR-Cpf1对PAM识别改变的结构基础
Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1.
作者信息
Nishimasu Hiroshi, Yamano Takashi, Gao Linyi, Zhang Feng, Ishitani Ryuichiro, 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; JST, PRESTO, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
出版信息
Mol Cell. 2017 Jul 6;67(1):139-147.e2. doi: 10.1016/j.molcel.2017.04.019. Epub 2017 Jun 6.
The RNA-guided Cpf1 nuclease cleaves double-stranded DNA targets complementary to the CRISPR RNA (crRNA), and it has been harnessed for genome editing technologies. Recently, Acidaminococcus sp. BV3L6 (AsCpf1) was engineered to recognize altered DNA sequences as the protospacer adjacent motif (PAM), thereby expanding the target range of Cpf1-mediated genome editing. Whereas wild-type AsCpf1 recognizes the TTTV PAM, the RVR (S542R/K548V/N552R) and RR (S542R/K607R) variants can efficiently recognize the TATV and TYCV PAMs, respectively. However, their PAM recognition mechanisms remained unknown. Here we present the 2.0 Å resolution crystal structures of the RVR and RR variants bound to a crRNA and its target DNA. The structures revealed that the RVR and RR variants primarily recognize the PAM-complementary nucleotides via the substituted residues. Our high-resolution structures delineated the altered PAM recognition mechanisms of the AsCpf1 variants, providing a basis for the further engineering of CRISPR-Cpf1.
RNA引导的Cpf1核酸酶可切割与CRISPR RNA（crRNA）互补的双链DNA靶点，并且已被用于基因组编辑技术。最近，人们对嗜酸栖热菌属菌株BV3L6（AsCpf1）进行了改造，使其能够识别经过改变的DNA序列作为原间隔序列临近基序（PAM），从而扩大了Cpf1介导的基因组编辑的靶标范围。野生型AsCpf1识别TTTV PAM，而RVR（S542R/K548V/N552R）和RR（S542R/K607R）变体则可分别有效识别TATV和TYCV PAM。然而，它们的PAM识别机制仍不清楚。在此，我们展示了与crRNA及其靶标DNA结合的RVR和RR变体的分辨率为2.0 Å的晶体结构。这些结构表明，RVR和RR变体主要通过取代的残基识别PAM互补核苷酸。我们的高分辨率结构描绘了AsCpf1变体改变后的PAM识别机制，为CRISPR-Cpf1的进一步工程改造提供了基础。
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