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• W2946870230 abstract "ABSTRACT Efficient targeted insertion of heterologous DNA into a genome remains a challenge in genome engineering. Recombinases that can introduce kilobase-sized DNA constructs require pre-existing recombination sites to be present in the genome and are difficult to reprogram to other loci. Genome insertion using current CRISPR-Cas methods relies on host DNA repair machinery, which is generally inefficient. Here, we describe a Cas-Transposon (CasTn) system for genomic insertions that uses a transposase fused to a catalytically-dead dCas9 nuclease to mediate programmable, site-specific transposition. CasTn combines the power of the Himar1 transposase, which inserts multi-kb DNA transposons into TA dinucleotides by a cut-and-paste mechanism, and the targeting capability of Cas9, which uses guide-RNAs to bind to specific DNA sequences. Using in vitro assays, we demonstrated that Himar-dCas9 proteins increased the frequency of transposon insertions at a single targeted TA dinucleotide by >300-fold compared to an untargeted transposase, and that site-specific transposition is dependent on target choice while robust to log-fold variations in protein and DNA concentrations. We then showed that Himar-dCas9 mediates site-specific transposition into a target plasmid in E. coli . This work provides CasTn as a new method for host-independent, programmable, targeted DNA insertions to expand the genomic engineering toolbox." @default.
• W2946870230 created "2019-06-07" @default.
• W2946870230 creator A5008137474 @default.
• W2946870230 creator A5077117890 @default.
• W2946870230 date "2019-06-03" @default.
• W2946870230 modified "2023-09-30" @default.
• W2946870230 title "An Engineered Cas-Transposon System for Programmable and Precise DNA Transpositions" @default.
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• W2946870230 doi "https://doi.org/10.1101/654996" @default.
• W2946870230 hasPublicationYear "2019" @default.
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