Fig. 2

Optimization of CRISPR-Cas-assisted and MMEJ-directed genomic editing. a Portion of MMEJ deletion at the targets and mutations at the targets or editing plasmids in the transformants carrying the editing plasmids. MMEJ: the genomes showed MMEJ mediated deletions covering the protospacers; protospacer mutations: point mutations observed at the protospacer sequences; others: mutations at the mini-CRISPR (Repeat–Spacer–Repeat cassette) on the editing plasmids, or at other genes resulted in loss of CRISPR function, etc. b PCR amplification and agarose gel analysis spacer excision at the mini-CRISPR of pS0672 (above) and pS0631 (below) plasmids on 1.5% agarose gel. Bands, marked by red arrows, indicated excision of the spacer. c Sequences and predicted structures of the natural and modified repeats of the “Repeat-Spacer-Repeat” cassette. The mutation at the 1st or the 2nd repeat was indicated. Specifically, the modified nucleotides were shown in red, and the truncated nucleotides were shown in grey. d Transformation efficiencies of the control plasmid and different interference plasmids. The significance was determined using a t test; p < 0.01 **, p < 0.001 ***. e Colony PCR screening of MMEJ mutants transformed with the interference plasmids carrying different Repeat sequences against the ZMO0672 gene. The lanes representing the transformants carrying MMEJ deletions were indicated by red arrows. f Portion of MMEJ deletion at the targets and mutations at the targets or editing plasmids in the transformants carrying the modified editing plasmids. Category “spacer excision” is included in the “others” in a