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Schematic representation of bacterium-based selection assay to isolate LbCas12a mutants with enhanced activity. E.coli BW25141:DE3 cells containing an inducible ccdB expression plasmid were transformed with LbCas12a variant library, which was programmed to cleave the reporter plasmid through a target site with TTTT PAM sequence. Active LbCas12a mutants with enhanced activity enable the clearance of reporter plasmid, thus avoiding the cell death upon the induction of ccdB expression with arabinose. LbCas12a plasmids from survived cells were extracted and used for subsequent round of selection. Four rounds of sequential selection were performed. Round 3 and 4 libraries were sequenced and used to calculate the enrichment of LbCas12a variants.

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