Fig. 4.

Fig. 4. Single-nucleotide level multiplex genome editing using 3'-end truncated sgRNAs. (A) A schematic diagram of simultaneous three-target editing. The 3'-end truncated sgRNA/Cas12f1 complexes cleave unedited target sequences while leaving single-nucleotide-substituted sequences intact. Therefore, cells survive only when editing events occur simultaneously at all three targets. (B) Multiplex single-nucleotide substitution in the galK, xylB, and srlD genes using 3'-end truncated sgRNAs. The efficiencies of editing galK ⁵⁰⁴A, xylB ⁶⁵²T, and srlD ³²⁸T using truncated sgRNAs were compared under two recovery conditions: 1 h at 37°C and 12 h at 17°C. (C) Sanger sequencing analysis of the single-nucleotide-edited cells. Four white colonies were randomly selected and the target sequences of the galK, xylB, and srlD genes were analyzed. The target nucleotides are highlighted in color and bold font.

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