Mg2+-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage

CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)–distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg²⁺ ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg²⁺-dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target.

CRISPR-Cas, a prokaryotic adaptive immune system, is a revolutionary tool for genome editing (1–6). Among the various types of the Cas systems, Cas12a (also known as Cpf1), class 2 type V-A CRISPR-Cas system, catalyzes double-stranded DNA (dsDNA) targets by utilizing single CRISPR RNA (crRNA) (7–10). The Cas12a-crRNA ribonucleoprotein (RNP) complex first identifies the dsDNA target via a T-rich protospacer-adjacent motif (PAM). Upon binding with cognate DNA, the Cas12a RNP unwinds the DNA via the formation of a crRNA-target strand (TS) heteroduplex and the simultaneous displacement of the nontarget strand (NTS) (a so-called R-loop structure) (11). Then, Cas12a generates double-strand DNA breaks with sticky ends by using a single RuvC nuclease domain in a sequential manner. Furthermore, in contrast to Cas9, Cas12a exhibits distinct features of pre-crRNA processing and indiscriminate single-stranded DNA cleavage activity (7, 12, 13). Owing to these unique features, CRISPR/Cas12a has been extensively utilized for the detection of nucleic acids as well as programmable genome editing (13–21).Meanwhile, recently reported base and prime editors, which accomplish targeted edits in a highly efficient manner, utilized a nickase form of CRISPR/Cas9 to reduce the frequency of undesired insertions and deletions (22–24). However, the distinct feature by which both strands of target DNA are cleaved by a single catalytic site of Cas12a has hampered the development of engineered Cas12a RNPs including an efficient nickase, resulting in a limited range of Cas12a application (25–27). Given the advantages of Cas12a, including its multiplexing capability using the intrinsic crRNA processing activity and fewer off-target effects compared to Cas9 (14, 15, 17, 28), the development of various engineered Cas12a RNPs is necessary to improve genome editing techniques. Although recently several studies have suggested the nickase form of Cas12a RNPs using alterations of crRNA (29) or mutations of protein residues (30, 31), existing nickase variants still have much room for enhancement of the nicking activity. In this regard, thorough understanding of the mechanisms that regulate the sequential cleavage reaction of dsDNA, beginning with the NTS and proceeding to the TS, by a single catalytic site in the Cas12a RuvC domain, is required. However, despite many recent biochemical and structural studies (30–40), a detailed mechanistic understanding of the way in which Cas12a uses its single catalytic site to completely break the double strand of the target DNA is still lacking.Here we perform single-molecule fluorescence assay to monitor conformational dynamics of the Cas12a ternary complex during TS cleavage following the initial cleavage of NTS of DNA. Recently, several groups have utilized similar methodological approaches to monitor the molecular interaction between Cas12a RNP and target DNA by using labeled target DNA and crRNA (35–37) and the interdomain dynamics of Cas12a protein by using labeled Cas12a (31, 41). Using this assay, here we identified the features of intermediates that form during conformational rearrangements in the TS cleavage reaction to complete dsDNA cleavage and revealed its underlying mechanism based on a kinetic analysis of the conformational dynamics. The results of our study suggest that Mg²⁺-mediated local DNA unwinding in the PAM-distal region is an essential prerequisite for the regulation of the sequential dsDNA cleavage reaction. This allosteric mechanism provides molecular insight into Cas12a engineering toward the development of Cas12a nickase.