GeneWeld: a method for efficient targeted integration directed by short homology

Choices for genome engineering and integration involve high efficiency but little or no target specificity or high specificity but low activity. Here, we describe a targeted integration strategy, called GeneWeld, and vector series for gene tagging, pGTag (plasmids for Gene Tagging), which promotes highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. A universal gRNA (UgRNA), with no predicted targets in zebrafish, pig, or human, efficiently targets the pGTag series donors at sites flanking cargo DNA, thus exposing homology engineered to match the sequence flanking a genomic double-strand DNA break. Our work demonstrates that in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 48 base pairs of homology directs precise and efficient knock-in. We expect our results and the accompanying vectors, protocols, and web interface for homology arm design will serve to streamline gene targeting and applications in CRISPR compatible systems.