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

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 precision gene targeting 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. Our vector series, pGTag (plasmids for Gene Tagging), contains
reporters flanked by a universal CRISPR sgRNA sequence to target double
strand breaks in vivo and expose 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. 1 kb long homology arms did not increase targeting efficiency. Our
system provides a straightforward and cost-effective approach for high
efficiency gene targeting applications in CRISPR and TALEN compatible
systems.