Mg <sup>2+</sup> -dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage
Heyjin Son, Jae-Il Park, In-Joo Hwang, Youngri Jung, Sangsu Bae, Sang-Hwa Lee
Abstract
Significance CRISPR-Cas12a has emerged as attractive molecular scissors alternative to Cas9 owing to its unique features including fewer off-target effects, an alternative protospacer-adjacent motif sequence, pre-CRISPR RNA processing activity, and indiscriminate single-stranded DNase activity. However, despite these advantages, Cas12a has not been well utilized as recently reported base and prime editors because it does not have complete nickase variants, unlike Cas9. In this study, we provide a thorough understanding of the mechanisms that govern the generation of complete double-stranded DNA breaks by the single catalytic site of Cas12a using single-molecule fluorescence assays to improve our ability to develop a rational design for more potently engineered Cas12a including the nickase form. This would extend the range of genome editing applications of Cas12a.