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.

RNA 引导的 Cpf1（也称为 Cas12a）核酸酶与 CRISPR RNA (crRNA) 结合，并切割与 crRNA 引导互补的双链 DNA 靶标。氨基酸球菌属的两个 Cpf1 直向同源物。 (AsCpf1) 和Lachnospiraceae 细菌(LbCpf1) 已被用于真核基因组编辑。 Cpf1 需要一个特定的核苷酸序列（称为原型间隔子相邻基序 (PAM)）来进行目标识别。除了规范的 TTTV PAM 之外，Cpf1 还可以识别次优的含 C PAM。在这里，我们报告了 LbCpf1 与 crRNA 及其靶 DNA 复合的四种晶体结构，其中包含 TTTA、TCTA、TCCA 或 CCCA 作为 PAM。这些结构表明，根据 PAM 序列，LbCpf1 会发生构象变化，与含有 PAM 的 DNA 双链体形成改变的相互作用，从而实现宽松的 PAM 识别。总的来说，目前的结构推进了我们对 Cpf1 家族核酸酶依赖 PAM 的、crRNA 引导的 DNA 切割的机制理解。