Adapting CRISPR-associated transposons for rapid and high-throughput reverse genetics.

ABSTRACT: CRISPR-associated transposons (CAST) use guide RNAs to direct their transposition and are being harnessed as tools for programmable genome engineering across diverse bacterial species. However, CAST systems have not been adapted for high-throughput genetic screening. Here, we present MultiCAST, a streamlined platform for rapid and scalable guide RNA-directed transposon insertion in bacteria. MultiCAST generates targeted insertions in a single step through conjugative delivery of conditionally replicative plasmids encoding the CAST enzymatic machinery and a selectable mini-transposon expressing the guide RNA. By leveraging the inserted guide sequence as a molecular barcode, MultiCAST enables pooled, high-throughput genetic screens using only amplicon sequencing. We identified factors that i

SUBMITTER: Basta DW 

PROVIDER: S-EPMC12636600 | biostudies-literature | 2025 Nov

REPOSITORIES: biostudies-literature