Size and sequence variation of the Lid of the Cas12a nuclease domain results in a nickase phenotype.

CRISPR-Cas12a nucleases typically induce double-strand breaks (DSBs) to initiate genome editing. However, reliance on DSBs risks chromosomal rearrangements and unintended insertions or deletions at off-target sites. Therefore, engineering nickase variants that cleave only a single DNA strand might enable precise editing platforms. We describe an unbiased in vivo screen in Escherichia coli to identify LbCas12a nickase variants. We targeted the RuvC Lid domain for mutagenesis, hypothesizing that altering this region would disrupt the coordination of strand cleavage. While the screen produced many nickase variants with targeted point substitutions (Lid1), it also serendipitously yielded a variant (Lid2) with an unprecedented Lid domain in terms of size and sequence. Biochemical characterization confirms that Lid2 robustly nicks the non-target strand (NTS) with minor cleavage activity observed for the target strand (TS), accumulating predominantly nicked substrates. Cryo-electron microscopy (cryo-EM) at 2.5 Å resolution reveals that, in Lid2, the native α-helical Lid is replaced, uncoupling the concerted cleavage mechanism. Specifically, the remodeled Lid appears to stabilize the NTS in the active site, whereas it hampers the conformational transitions required for subsequent TS cleavage. This robust nickase-phenotype of this Cas12a variant expands the CRISPR-Cas toolbox and offers a new component for precise genome editing applications.