Efficient targeted integration directed by short homology in zebrafish and mammalian cells

• Main Authors: Wesley A Wierson, Jordan M Welker, Maira P Almeida, Carla M Mann, Dennis A Webster, Melanie E Torrie, Trevor J Weiss, Sekhar Kambakam, Macy K Vollbrecht, Merrina Lan, Kenna C McKeighan, Jacklyn Levey, Zhitao Ming, Alec Wehmeier, Christopher S Mikelson, Jeffrey A Haltom, Kristen M Kwan, Chi-Bin Chien, Darius Balciunas, Stephen C Ekker, Karl J Clark, Beau R Webber, Branden S Moriarity, Stacy L Solin, Daniel F Carlson, Drena L Dobbs, Maura McGrail, Jeffrey Essner
• Format: Article
• Language: English
• Published: eLife Sciences Publications Ltd 2020-05-01
• Series: eLife
• Subjects: CRISPR/Cas9; knock-in; targeted integration; human; pig fibroblasts; end joining
• Online Access: https://elifesciences.org/articles/53968

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.