Fig. 3.

Fig. 3. Multiplex three-target genome editing. (A) Plasmid expressing three sgRNAs (targeting galK, xylB, and srlD, respectively). The triple sgRNA plasmid was designed to avoid the loss of sgRNAs resulting from recombination between repetitive scaffold sequences. Each sgRNA cassette expresses galK-, xylB-, and srlD-targeting sgRNAs. (B) Multiplex 4 nt substitution in the galK, xylB, and srlD genes. The efficiencies of editing galK ⁵⁰⁴ATCA, xylB ⁶⁴⁹AACT, and srlD ³²³GTTA were compared under two recovery conditions: 1 hour at 37°C and 12 hours at 17°C. (C) Multiplex single-nucleotide substitution in the galK, xylB, and srlD genes. The efficiency of editing galK ⁵⁰⁴A, xylB ⁶⁵²T, and srlD ³²⁸T was compared under two recovery conditions: 1 h at 37°C and 12 h at 17°C.

© J. Microbiol. Biotechnol.