Fig. 5

Large fragment deletion via endogenous CRISPR interference and MMEJ repair. a Schematic of simultaneous targeting by the self-targeting plasmid pS1807-15 carrying “Repeat-Spacer(1807)-Repeat-Spacer(1815)-Repeat” expression cassette against the protospacers at ZMO1807 and ZMO1815 gene loci. The PAM sequences for the protospacers and the primers used to amplify the region between two protospacers are indicated. The distance between two protospacers and the primers are shown, respectively. b Transformation efficiency of pS1807-15 relative to the empty plasmid pEZ15Asp. Error bars represent the SD of three independent experiments. The significance was determined using a t test; p < 0.001 ***. c Agarose gel analysis of the PCR products covering the regions between two protospacers using the primers 1807-test-F and 1822-test-R from three randomly selected single colonies. The sizes of the PCR products are indicated. M, DNA size marker. d Schematic of the MMEJ-mediated deletion regions. The distances between the microhomology repeats to the protospacers and the deletion sizes are indicated, respectively. e Growth curves of Z. mobilis ZM4mrr strain and the large size deletion strain 1 in the RM medium