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. 2022 Dec 20;50(22):12689–12701. doi: 10.1093/nar/gkac1192

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© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Enhancing the cis-cleavage activity of mut2 by directed evolution. (A) Schematic illustration of the positive selection system used for directed evolution of mut2 LbCas12a. (B) Generation of 3 beneficial mutants from mut2 (starter) by directed evolution. Upper panel showing the region (R) divisions of LbCas12a protein used for error-prone polymerase chain reaction (PCR) mutagenesis. Lower panel showing the mutants selected followed each round of mutagenesis. Three beneficial mutants (named mut2A, mut2B, and mut2C) were generated from two rounds of selection. The mutations in each variant are listed. (C) Snapshots of LbCas12a protein structure (PDB ID: 5XUS) highlighting the locations of the beneficial mutations obtained from directed evolution.