Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1

The RNA-guided Cpf1 (also known as Cas12a) nuclease associates with a CRISPR RNA (crRNA) and cleaves the double-stranded DNA target complementary to the crRNA guide. The two Cpf1 orthologs from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) have been harnessed for eukaryotic genome editing. Cpf1 requires a specific nucleotide sequence, called a protospacer adjacent motif (PAM), for target recognition. Besides the canonical TTTV PAM, Cpf1 recognizes suboptimal C-containing PAMs. Here, we report four crystal structures of LbCpf1 in complex with the crRNA and its target DNA containing either TTTA, TCTA, TCCA, or CCCA as the PAM. These structures revealed that, depending on the PAM sequences, LbCpf1 undergoes conformational changes to form altered interactions with the PAM-containing DNA duplexes, thereby achieving the relaxed PAM recognition. Collectively, the present structures advance our mechanistic understanding of the PAM-dependent, crRNA-guided DNA cleavage by the Cpf1 family nucleases. [Display omitted] •Cpf1 recognizes canonical TTTV and non-canonical C-containing PAMs•Crystal structures of L. bacterium Cpf1 in complex with the crRNA and its target DNA•PAM recognition mechanism is highly conserved among the Cpf1 enzymes•PAM-interacting domain displacement plays roles in the relaxed PAM recognition Yamano et al. report the crystal structures of L. bacterium Cpf1 bound to the crRNA and its target DNA with distinct PAMs, providing insights into the recognition mechanism of the optimal TTTV and suboptimal C-containing PAMs.