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

Authors :
Jacklyn Levey
Merrina Lan
Carla M. Mann
Maura McGrail
Maira P. Almeida
Beau R. Webber
Kristen M. Kwan
Dennis A. Webster
Trevor J. Weiss
Jeffrey J. Essner
Darius Balciunas
Wesley A. Wierson
Karl J. Clark
Zhitao Ming
Jeffrey A. Haltom
Alec Wehmeier
Stephen C. Ekker
Melanie E. Torrie
Christopher S. Mikelson
Stacy L. Solin
Sekhar Kambakam
Daniel F. Carlson
Macy K. Vollbrecht
Branden S. Moriarity
Jordan M. Welker
Chi Bin Chien
Drena Dobbs
Kenna C. McKeighan
Source :
eLife, Cambridge : eLife Sciences Publications, Ltd, 2020, vol. 9, art. no. e53968, p. [1-25], eLife, Vol 9 (2020), eLife
Publication Year :
2020

Abstract

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.

Details

Language :
English
ISSN :
2050084X
Database :
OpenAIRE
Journal :
eLife, Cambridge : eLife Sciences Publications, Ltd, 2020, vol. 9, art. no. e53968, p. [1-25], eLife, Vol 9 (2020), eLife
Accession number :
edsair.doi.dedup.....bfdaa4c2ba4444b21a6e2fac41b3b74c