Campus Units

Genetics, Development and Cell Biology

Document Type

Article

Publication Version

Published Version

Publication Date

2020

Journal or Book Title

eLife

Volume

9

First Page

e53968

DOI

10.7554/eLife.53968

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.

Comments

This article is published as Wierson, Wesley A., Jordan M. Welker, Maira P. Almeida, Carla M. Mann, Dennis A. Webster, Melanie E. Torrie, Trevor J. Weiss et al. "Efficient targeted integration directed by short homology in zebrafish and mammalian cells." Elife 9 (2020): e53968. doi: 10.7554/eLife.53968.

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

Copyright Owner

Wierson et al.

Language

en

File Format

application/pdf

Share

COinS