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GeneWeld: a method for efficient targeted integration directed by short homology
  • Wesley A. Wierson, Iowa State University
  • Jordan M. Welker, Iowa State University
  • Maira P. Almeida, Iowa State University
  • Carla A. Mann, Iowa State University
  • Dennis A. Webster, Recombinetics, Inc.
  • Melanie E. Torrie, Iowa State University
  • Trevor J. Weiss, Iowa State University
  • Macy K. Vollbrecht, Recombinetics, Inc.
  • Merrina Lan, Iowa State University
  • Kenna C. McKeighan, Iowa State University
  • Zhitao MIng, Iowa State University
  • Alec Wehmeier, Iowa State University
  • Christopher S. Mikelson, Iowa State University
  • Jeffrey A. Haltom, Iowa State University
  • Kristen M. Kwan, University of Utah School of Medicine
  • Chi-­Bin Chien, University of Utah Medical Center
  • Darius Balciunas, Temple University
  • Stephen C. Ekker, Mayo Clinic
  • Karl J. Clark, Mayo Clinic
  • Beau R. Webber, University of Minnesota - Twin Cities
  • Branden Moriarity, University of Minnesota - Twin Cities
  • Staci L. Solin, Recombinetics, Inc.
  • Daniel F. Carlson, Recombinetics, Inc.
  • Drena L. Dobbs, Iowa State University
  • Maura McGrail, Iowa State University
  • Jeffrey J. Essner, Iowa State University
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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.


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

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Wesley A. Wierson, Jordan M. Welker, Maira P. Almeida, Carla A. Mann, et al.. "GeneWeld: a method for efficient targeted integration directed by short homology" bioRxiv (2018)
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