Fig. 1

Establishment of a gene deletion method based on CRISPR-Cas system and MMEJ pathway in Z. mobilis. a Experimental paradigm for the gene deletion method. A self-targeting plasmid pS0626 (0626 relative to the target gene ZMO0626) carrying the expression cassette of “Repeat-Spacer-Repeat”, where the spacer matches the target DNA sequence followed a NCC PAM in ZMO0626 as an example, is electroporated into Z. mobilis ZM4mrr cells. The guide RNA transcribed and processed from the “Repeat-Spacer-Repeat” cassette guides DNA cleavage at the target by the endogenous subtype I–F surveillance complex, and the cleaved DNA is subsequently repaired through MMEJ pathway. b Relative transformation efficiency of each self-interference plasmid against ZMO0626, ZMO0631, ZMO0672, ZMO1063, ZMO1404, ZMO1807, ZMO1815 and ZMO1822 genes, relative to the empty plasmid pEZ15Asp. Error bars represent the SD of three independent experiments. The significance was determined using a t test; p < 0.05 *, p < 0.01 **, p < 0.001 ***. c MMEJ efficiencies of the randomly selected colonies carrying each self-targeting plasmids. Error bars represent the SD of three independent experiments. d GC content of the protospacers affected the MMEJ repair efficiency after CRISPR interference. The locations of five protospacers on ZMO0672 gene with different GC content were shown above, and GC content, transformation efficiency, MMEJ efficiency were shown below