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.
journal_name
Elifejournal_title
eLifeauthors
Wierson WA,Welker 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,Wedoi
10.7554/eLife.53968subject
Has Abstractpub_date
2020-05-15 00:00:00issn
2050-084Xpii
53968journal_volume
9pub_type
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