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

Abstract

Summary 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 the 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.

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“…6a). Success using this suboptimal CTTC PAM was previously demonstrated 45 . Using S10, we delivered Cas12a RNPs and achieved editing in large ( Fig.…”

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“…The REC lobe of Cas9 comprises REC1 and REC2 domains, whereas the NUC lobe comprises the PAM‐interacting (PI), RuvC, and HNH domains (Nishimasu et al ., ). Cas12a family proteins are generally smaller than most of the Cas9 orthologs because they lack an HNH domain (Yamano et al ., ). The CRISPR/Cas12a system was first used for targeted genome editing in mammalian cells (Kim et al ., ), and has since been successfully used for genome editing in plants, such as Arabidopsis thaliana (Tang et al ., ), rice ( Oryza sativa ) (Hu et al ., ; Tang et al ., ; Wang et al ., ; Xu et al ., ; Li et al ., ), soybean ( Glycine max ) (Kim et al ., ), tobacco ( Nicotiana tabacum ) (Kim et al ., ), maize ( Zea mays ) (Lee et al ., ), and the unicellular green alga ( Chlamydomonas reinhardtii ) (Ferenczi et al ., ).…”

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“…In contrast, different molecular mechanisms in delivering DSBs by Cas12a proteins in type V CRISPR‐Cas system have been revealed by recent studies on the crystal structures of Cas12a‐crRNA‐DNA complexes, such as AsCas12a from Acidaminococcus sp ., FnCas12a from Francisella novicida , and LbCas12a from Lachnospiraceae bacterium . Cas12a proteins have a bilobed molecular architecture as Cas9 proteins, consisting of a recognition lobe (REC) and a nuclease lobe (NUC) (Figure c).…”

“…For instance, LbCas12a and AsCas12a prefer a PAM sequence as 5′‐TTTV‐3′, while FnCas12a adopts a PAM site as 5′‐TTV‐3′ (Figure e) . In addition, noncanonical PAMs are also acceptable, but the targeting efficiencies are substantially reduced …”