SNAP output for PDB entry 5xh7 [SNAP web server]

PDB-id

5xh7; SNAP-derived features in text and JSON formats; DNAproDB

Class

hydrolase-RNA-DNA

Method

X-ray (2.0 Å)

Summary

Crystal structure of the acidaminococcus sp. bv3l6 cpf1 rr variant in complex with crrna and target DNA (tcca pam)

Reference

Nishimasu H, Yamano T, Gao L, Zhang F, Ishitani R, Nureki O (2017): "Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1." Mol. Cell, 67, 139-147.e2. doi: 10.1016/j.molcel.2017.04.019.

Abstract

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.