GeneWeld: a method for efficient targeted integration directed by short homology

Date
2018-10-03
Authors
Wierson, Wesley
Welker, Jordan
Almeida, Maira
Mann, Carla
Webster, Dennis
Torrie, Melanie
Weiss, Trevor
Vollbrecht, Macy
Lan, Merrina
McKeighan, Kenna
MIng, Zhitao
Wehmeier, Alec
Mikelson, Christopher
Haltom, Jeffrey
Kwan, Kristen
Chien, Chi-­Bin
Balciunas, Darius
Ekker, Stephen
Clark, Karl
Webber, Beau
Moriarity, Branden
Solin, Staci
Carlson, Daniel
Dobbs, Drena
McGrail, Maura
Essner, Jeffrey
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Abstract

Choices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand break in vivo. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR compatible systems.

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This is a pre-print made available through bioRxiv, doi: 10.1101/431627.

Keywords
CRISPR/Cas9, knock-­in, homology mediated-­end joining, targeted integration, zebrafish, pig fibroblasts, human K-­562 cells
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