Multiplex Single-Nucleotide Microbial Genome Editing Achieved by CRISPR-Cas9 Using 5'-End-Truncated sgRNAs.

ABSTRACT: Multiplex genome editing with CRISPR-Cas9 offers a cost-effective solution for time and labor savings. However, achieving high accuracy remains a challenge. In an Escherichia coli model system, we achieved highly efficient single-nucleotide level simultaneous editing of the galK and xylB genes using the 5'-end-truncated single-molecular guide RNA (sgRNA) method. Furthermore, we successfully demonstrated the simultaneous editing of three genes (galK, xylB, and srlD) at single-nucleotide resolution. To showcase practical application, we targeted the cI⁸⁵⁷ and ilvG genes in the genome of E. coli. While untruncated sgRNAs failed to produce any edited cells, the use of truncated sgRNAs allowed us to achieve simulta

SUBMITTER: Lim SR 

PROVIDER: S-EPMC10368013 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature