Fig. 2

CRISPR–Cas12a system as a new robust genome editing tool in M. thermophila. a Schematic illustration of the mutagenesis of amdS for measuring Cas12a-mediated DNA cleavage in M. thermophila. Positive transformants were selected on plates containing 2 mg mL⁻¹ FAA. b Indel patterns at the amdS target locus of FAA-resistant transformants. The number on the right of each sequence is the indel length (−, deletion). Blue, crRNA base-pairing site; red, PAM sequences; WT, wild-type sequence. c Schematic illustration of CRISPR–Cas12a-donor DNA-mediated deletion of the target gene cre-1 based on homology-directed repair (HDR). Twenty transformants were selected and verified by PCR analysis. The expected length of the deletion mutant was 1.9 kb, whereas that of the wild-type strain (WT), which was the negative control, was 1.0 kb (rightmost lane). Heterokaryotic transformants showed two PCR bands (both of wild-type and knockout). The symbol of star indicated deletion mutant