3 years ago

Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1

Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1
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.

Graphical abstract

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Teaser

Variants of AsCpf1 with altered PAM specificities have been derived. Nishimasu et al. determined the high-resolution crystal structures of two of these variants bound to a crRNA and its target DNA, thereby explaining their altered PAM specificities.

Publisher URL: www.sciencedirect.com/science

DOI: S1097276517302757

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