New Target Gene Screening Using Shortened and Random sgRNA Libraries in Microbial CRISPR Interference.

ABSTRACT: CRISPR interference (CRISPRi) screening has been used for identification of target genes related to specific phenotypes using single-molecular guide RNA (sgRNA) libraries. In CRISPRi screening, the sizes of random sgRNA libraries contained with the original target recognition sequences are large (∼10¹²). Here, we demonstrated that the length of the target recognition sequence (TRS) can be shortened in sgRNAs from the original 20 nucleotides (N₂₀) to 9 nucleotides (N₉) that is still sufficient for dCas9 to repress target genes in the xylose operon of Escherichia coli, regardless of binding to a promoter or open reading frame region. Based on the results, we constructed random sgRNA plasmid libraries with 5'-shortened TRS lengths, and identified xylose

SUBMITTER: Jeong SH 

PROVIDER: S-EPMC10028695 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature