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. 2020 Jan 28;11(1):e02364-19. doi: 10.1128/mBio.02364-19

FIG 2.

FIG 2

CRISPR-Cas12a-assisted NHEJ genome editing in M. marinum. (A) Genome-editing efficiency in the presence of CRISPR-Cas12a cleavage in M. marinum. Plasmids pYC1103, pYC1178, and pYC1521, expressing whiB6, recD, and nrgA-targeting crRNA, respectively, were transformed into M. marinum with the Cas12a-expressing plasmid pMV261-Cas12a. (B) Genome-editing efficiency of whiB6 with CRISPR-Cas12a cleavage in wild-type M. marinum and its derivatives. The whiB6 crRNA plasmid (pYC1103) was transformed into the wild type and its derivatives. Transformation efficiency was defined as the total number of CFU generated per transformation. Editing efficiency was calculated as the ratio of the number of edited events to the total number of colonies analyzed by PCR and sequencing. Survival was calculated by comparing the number of transformants to the number of control transformants carrying the empty vector. (A, B) Bars represent mean values ± standard deviations from two independent experiments. (C) Deletion length distribution of the indicated genes resulting from CRISPR-Cas12-assisted genome editing. Bars represent the median deletion size for each strain.