Fig. 1

High-throughput characterization of DNA cleavage activities of LbCas12a point mutations in E. coli. A 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. B Design of a saturation mutagenesis library for LbCas12a. Every codon of LbCas12a was randomized with NNK degenerate primers by nicking mutagenesis [10]. Importantly, each member of plasmid library only contains one codon change at a time, which was verified by Sanger sequencing of 24 individual plasmids from the library. C Enrichment scores for ~ 9000 LbCas12a point mutations over the last round of selection (round 4). Synonymous variants without changes on the protein sequences were colored in red. Variants with a minimal of 50 counts were included in this analysis. D Enrichment scores for 5 selected positions of LbCas12a that have been evaluated prior to this study