Targeted integration in rat and mouse embryos with zinc-finger nucleases

Abstract

Gene targeting is indispensible for reverse genetics and the generation of animal models of disease. The mouse has become the most commonly used animal model system owing to the success of embryonic stem cell-based targeting technology, whereas other mammalian species lack convenient tools for genome modification. Recently, microinjection of engineered zinc-finger nucleases (ZFNs) in embryos was used to generate gene knockouts in the rat and the mouse by introducing nonhomologous end joining (NHEJ)-mediated deletions or insertions at the target site. Here we use ZFN technology in embryos to introduce sequence-specific modifications (knock-ins) by means of homologous recombination in Sprague Dawley and Long-Evans hooded rats and FVB mice. This approach enables precise genome engineering to generate modifications such as point mutations, accurate insertions and deletions, and conditional knockouts and knock-ins. The same strategy can potentially be applied to many other species for which genetic engineering tools are needed.

Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases

“…In contrast, the rate of correctly ARTICLE recombined clones was increased if both TALENs and the targeting construct were transfected together. From these observations we conclude that the construct integration rate in oocytes, and the resulting frequency of homologously mutated animals, although slightly lower than reported (for example, for the ROSA26 locus using zinc-finger nucleases), is still within an acceptable range given the size of the targeting construct [23][24][25][26] . We observed TALEN-mediated induction of NHEJ in 25.4% of the offspring independent of targeting construct integration.…”

Heritable gene-targeting with gRNA/Cas9 in rats

“…In addition to ZFN, new genomeediting tools such as transcription activator-like effector nuclease (TALEN) [5] and the clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPRassociated protein (Cas) system [6] have offered a rapid and efficient means of genome modification in many species. Technologies of ZFN and TALEN have made genetargeting in the rat genome more convenient and practical [7][8][9]. The CRISPR/Cas system is the most recently developed technology for targeted genome modification in mammalian cells, bacteria, zebrafish and mice [10][11][12].…”

Generating rats with conditional alleles using CRISPR/Cas9

“…Several genome-editing technologies, such as zinc-finger nucleases (ZFNs) [1][2], transcription activator-like effector nucleases (TALENs) [3] and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) system [4][5], have been used to produce knockout rat models by generating DNA double-strand breaks (DSBs) followed by non-homologous end joining (NHEJ)-mediated repair. However, the simple knockout strategies have limits for studying genes that are critical for embryogenesis.…”