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.

sgRNA targeted mutagenesis efficiency was determined by measuring the frequency of indel mutations at the target site. Individual embryos were injected with 150 pg Cas9 mRNA and 25 pg sgRNA. PCR amplicons over the cut site were first examined for mutagenesis (or a smear) on a 2-3% agarose gels in 1X TAE.  For the noto exon 1, cx43.4 exon 2, esama exon 2, msna exon 6, rb1 exon 2, exon 4 and exon 25, aqp1a1 exon 1 and aqp8a1 exon 1 sgRNAs, at 3 dpf 5 embryos were pooled in 50 ul of 50 mM NaOH, and 2 ul used as template for PCR. Barcoded libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645L) for MiSeq 250 bp single read sequencing at the University of Kansas Genome Sequencing Core. Indel analysis of MiSeq reads was performed using Cas-Analyzer at CRISPR RGEN Tools (http://www.rgenome.net/cas-analyzer/#!).