Fig. 3

CRISPR–Cas12a-mediated multiplex genome editing in M. thermophila. a Schematic illustration of the simultaneous deletion of three target genes, cre-1, res-1 and gh1-1, using the CRISPR–Cas12a system with pooled single-crRNA cassettes. b Schematic illustration of the simultaneous deletion of three target genes, cre-1, res-1 and gh1-1, using the CRISPR–Cas12a system with crRNA array1 expressing cassette in the order cre1–res1–gh1-1. c Once Cas12a nuclease introduces the sequence-specific double-stranded DNA break, homology-directed repair (HDR) mediate precise gene deletion using donor DNA template at the cleavage locus of cre-1, res-1 and gh1-1, respectively. DR, 19 nt direct repeat; U6p, MtU6 promoter; Ptef1, Tef1 promoter; NLS, nuclear localization signal; TtrpC, trpC Terminator