Fig. 5.

Fig. 5. Multiplex genome editing using CRISPR-Cas9 and low-temperature recovery. (A) The 5'-end truncated sgRNAs targeting the galK, xylB, and srlD genes. These 5'-end truncated sgRNAs form a complex with Cas9, facilitating the recognition and cleavage of the targets. If a single-nucleotide substitution occurs (galK ⁵⁰⁴A, xylB ⁶⁵²T, and srlD ³²⁸T), the target is not cleaved. (B) CRISPR-Cas9-mediated multiplex single-nucleotide substitution in the galK, xylB, and srlD genes using 5'-end truncated sgRNAs and low-temperature recovery. The efficiencies of editing galK ⁵⁰⁴A, xylB ⁶⁵²T, and srlD ³²⁸T using the 5'-end 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. An undesired mutation is indicated by a yellow box.

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