5XH6

Crystal structure of the Acidaminococcus sp. BV3L6 Cpf1 RVR variant in complex with crRNA and target DNA (TATA PAM)

Summary for 5XH6

• Entry DOI: 10.2210/pdb5xh6/pdb
• Related: 5XH7
• Descriptor: CRISPR-associated endonuclease Cpf1, crRNA, Target DNA strand, ... (8 entities in total)
• Functional Keywords: nuclease, hydrolase-rna-dna complex, hydrolase/rna/dna
• Biological source: Acidaminococcus sp. (strain BV3L6); More
• Total number of polymer chains: 4
• Total formula weight: 180246.86

Authors

Nishimasu, H.,Yamano, T.,Ishitani, R.,Nureki, O. (deposition date: 2017-04-19, release date: 2017-06-14, Last modification date: 2023-11-22)

Primary citation

Nishimasu, H.,Yamano, T.,Gao, L.,Zhang, F.,Ishitani, R.,Nureki, O.
Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1
Mol. Cell, 67:139-147.e2, 2017

PubMed 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.

PubMed: 28595896
DOI: 10.1016/j.molcel.2017.04.019

Experimental method

X-RAY DIFFRACTION (2 Å)