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A universal system for streamlined genome integrations with CRISPR-associated transposases

Wang, Megan and Sanfiorenzo, Charles and Zhang, Raymond J. and Wang, Kaihang (2022) A universal system for streamlined genome integrations with CRISPR-associated transposases. . (Unpublished)

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Genome engineering tools in bacteria are limited by their targeting abilities, cargo size capacities, and integration efficiencies. Programmable Cas-directed transposons have been shown to bypass these constraints; however, genome integrations with these Cas-directed transposons require a cargo plasmid carrying the desired DNA payload flanked by directed repeat transposon arms. This cloning pre-requisite significantly hinders the modularity and streamlining capabilities of Cas-directed transposon systems, diminishing their utility for genome engineering. Here, we present a system that can robustly integrate a linear DNA payload into the genome of E. coli by employing a Type I-F CRISPR-associated transposon from Vibrio cholerae. This system bypasses the traditional limiting factors of Cas-directed transposons by leveraging oligonucleotide design and nested polymerase chain reactions to reconstitute the transposon arms onto a designated cargo. Our findings demonstrate that this programmable linear integration method has high efficiencies in integrating large DNA payloads across multiple genomic loci.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription Paper Information ItemCode
Wang, Megan0000-0001-8845-4936
Wang, Kaihang0000-0001-7657-8755
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Work was supported by the Shurl and Kay Curci Foundation (grant no. 108625, K.W.) and the NIH (DP2-GM140937, K.W.). R.J.Z is supported by the National Science Foundation Graduate Research Fellowship (grant no. 1745301). We thank Igor Antoshechkin from the Millard and Muriel Jacobs Genetics and Genomics Laboratory for sequencing library preparations and Illumina amplicon NGS. This work was supported by the Shurl and Kay Curci Foundation (grant no. 108625) and the NIH (DP2-GM140937). Author Contributions. M.W. and C.S. conceived and designed this research. R.J.Z and M.W. cloned the plasmids used in this study. M.W. performed laboratory experiments and analyzed data. M.W. performed phenotyping analyses and R.J.Z. performed PCR-based genotype analyses. M.W. analyzed and graphed NGS data. M.W., C.S., and R.J.Z. wrote the paper with input from all authors. K.W. provided insightful guidance for the work. Data availability: we declare that the data supporting the findings of this study are available within the paper and its Supporting Information. NGS analysis pipelines can be found at Raw sequencing data produced and used in this study are available from the National Center for Biotechnology Information (NCBI) under SRA accession no. PRJNA835982. The authors have declared no competing interest.
Group:Millard and Muriel Jacobs Genetics and Genomics Laboratory
Funding AgencyGrant Number
Shurl and Kay Curci Foundation108625
NSF Graduate Research FellowshipDGE-1745301
Subject Keywords:CRISPR-associated transposons, genome engineering, large DNA insert, Lambda-Red, genome integration, synthetic biology
Record Number:CaltechAUTHORS:20220601-257848000
Persistent URL:
Official Citation:A universal system for streamlined genome integrations with CRISPR-associated transposases Megan Wang, Charles Sanfiorenzo, Raymond J Zhang, Kaihang Wang bioRxiv 2022.05.30.494051; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:114996
Deposited By: George Porter
Deposited On:01 Jun 2022 22:07
Last Modified:01 Jun 2022 22:07

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