Crystal structure of Cpf1 in complex with guide RNA and target DNA

By T. Yamano, H. Nishimasu, Bernd Zetsche, H. Hirano, I. Slaymaker, Yinqing Li, I. Fedorova, Takanori Nakane, Kira S. Makarova, Eugene V. Koonin and 3 more. Cell, 2016

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Cpf1 is an RNA-guided endonuclease of a type V CRISPR-Cas system that has been recently harnessed for genome editing. Here, we report the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with the guide RNA and its target DNA, at 2.8 Å resolution. AsCpf1 adopts a bilobed architecture, with the RNA–DNA heteroduplex bound inside the central channel. The structural comparison of AsCpf1 with Cas9, a type II CRISPR-Cas nuclease, reveals both striking similarity and major differences, thereby explaining their distinct functionalities. AsCpf1 contains the RuvC domain and a putative novel nuclease domain, which are responsible for the cleavage of the non-target and target strands, respectively, and jointly generate staggered DNA double-strand breaks. AsCpf1 recognizes the 5′-TTTN-3′ protospacer adjacent motif by base and shape readout mechanisms. Our findings provide mechanistic insights into RNA-guided DNA cleavage by Cpf1, and establish a framework for rational engineering of the CRISPR-Cpf1 toolbox.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/48717cc0418c1cc43b0e4bef88379bcfd22e5650

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Citations in the corpus

New CRISPR-Cas systems from uncultivated microbes (2016, methodology): “High scoring HHpred hits were used to infer domain architecture based on comparison to solved crystal structures 50,51 , and secondary structure that was predicted by JPred4 52 .”
CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA (2018, background): “The RuvC catalytic pocket is consistently exposed in the bilobed structure of the ternary Cas12a complexes 8 (Supplementary information, Figure S11f) but not in the structurally dynamic Cas12a monomer 9 nor in the triangle-shaped binary structure 9 (Supplementary information, Figure S11g), allowing…”
CRISPR-Cas12a: Functional overview and applications (2020, background): “The PAM for LbCas12a and AsCas12a has a sequence of 50-TTTN-3' and for FnCas12a a sequence of 50-TTN-30 and is situated upstream of the 5'end of the non-target strand [26,31,34].”
The Biology of CRISPR-Cas: Backward and Forward. (2018, positive): “…to type II systems, which utilize diverse PAMs (Fonfara et al., 2014) that are located on the non-target strand, Cas12 proteins recognize the PAM on both DNA strands, with the non-target PAM sequence being T-rich (Fonfara et al., 2016; Yamano et al., 2016; Yang et al., 2016; Zetsche et al., 2015).”
Multiplex gene editing by CRISPR-Cpf1 through autonomous processing of a single crRNA array (2016, background): “, 2016), we identified five conserved amino acid residues close to the 5’ end of the DR that are likely to disrupt array processing and mutated them to alanine (H800, K809, K860, F864, and R790) (Yamano et al., 2016).”