Chunlai Chen’s lab reported how conformational dynamics of Cas12a governs DNA cleavage

2019-08-09 11:58:07

On August 6th 2019, the single-molecule biophysics group led by Dr. Chunlai Chen from School of Life Sciences, Tsinghua University published a research article entitled “Conformational dynamics and cleavage sites of Cas12a are modulated by complementarity between crRNA and DNA” in iScience. In this study, researchers applied single-molecule fluorescence tools to reveal conformational dynamics of Cas12a/crRNA/dsDNA ternary complexes governing DNA cleavage, which provided mechanistic insights regarding how high specificity, well-defined cleavage order, and multi-cleavage sites of Cas12a were achieved.

Cas12a protein, also known as Cpf1, displays its own unique features and serves as an alternative and complementation to Cas9 for genome editing and engineering. Although Cas12a has attracted great attention and been widely used, several fundamental questions, including how its nuclease activity is activated, how it uses a single RuvC endonuclease domain to cleave non-target strand and target strands sequentially, and how it is able to target different cleavage sites on dsDNA, are not fully resolved. The Chen group used single-molecule fluorescence resonance energy transfer (smFRET) to quantitatively analyze how conformational dynamics of Cas12a/crRNA/dsDNA ternary complexes are modulated by crRNA-DNA mismatches, pre-unwound dsDNAs, and pre-cleaved dsDNAs. Four distinctive conformational states were identified, with which a quantitative kinetic scheme was established to describe reaction pathways of Cas12a ternary complexes. When Cas12a/crRNA binary complex recognizes its target, ternary complex is formed as the low-FRET state, an intermediate state containing partially formed R-loop. Extension of R-loop to the PAM-distal end causes further interactions between Cas12a and the heteroduplex, which trigger conformational changes of Cas12a to activate its nuclease center and to adopt the non-target strand pre-cleavage conformational state identified as the medium-FRET state. Once the non-target strand is cleaved, target strand pre-cleavage states are greatly stabilized and become long-lived states (two high-FRET states). The extent of complementarity between crRNA and target strand modulates the relative stabilities of S3 and S4, which eventually causes different patterns of cleaved fragments.

The key aspects of their discoveries are summarized below. 1) The highly dynamic nature of Cas12a complexes, including their reversible formation, disassembly and transition between different conformational states, is likely to be one of the key aspects contributing to their high specificity. 2) The non-target strand is cleaved when its cleavage sites are released from DNA duplex after DNase activation of Cas12a, which serves as an additional important checkpoint to license dsDNA cleavage. 3) Cleaved non-target strand stabilizes target strand pre-cleavage states to permit subsequent cleavage and to ensure two DNA strands cleaved in a well-defined order. 4) The extent of complementarity between crRNA and DNA modulates relative stabilities of target strand pre-cleavage states targeting different cleavage sites. Discoveries made by the Chen group provide insights to fully elucidate working mechanisms of Cas12a and to optimize it for genome engineering.

This project was supported by funds from the National Natural Science Foundation of China, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology and Beijing Frontier Research Center for Biological Structure. The authors would like to thank Dr. Zhiwei Huang from Harbin Institute of Technology for providing plasmid to express LbCas12a. Lujia Zhang, a PhD student from School of Life Sciences, Tsinghua University is the first author. Dr. Chunlai Chen is the corresponding author of this article.

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