Efficient targeted integration directed by short homology in zebrafish and mammalian cells
Genre
Journal ArticleDate
2020-05-01Author
Wierson, WAWelker, JM
Almeida, MP
Mann, CM
Webster, DA
Torrie, ME
Weiss, TJ
Kambakam, S
Vollbrecht, MK
Lan, M
McKeighan, KC
Levey, J
Ming, Z
Wehmeier, A
Mikelson, CS
Haltom, JA
Kwan, KM
Chien, CB
Balciunas, D
Ekker, SC
Clark, KJ
Webber, BR
Moriarity, BS
Solin, SL
Carlson, DF
Dobbs, DL
McGrail, M
Essner, J
Subject
CRISPR/Cas9developmental biology
end joining
genetics
genomics
human
knock-in
pig fibroblasts
targeted integration
zebrafish
Permanent link to this record
http://hdl.handle.net/20.500.12613/4253
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10.7554/eLife.53968Abstract
© Wierson et al. 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.Citation to related work
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http://dx.doi.org/10.34944/dspace/4235