High-efficiency And Heritable Gene Targeting In Mouse And Rat Using TALEN And CRISPR/Cas System

Targeted-knockout animal models play an important role in understanding physiological and pathological mechanism as well as the development of new drugs. Since the physiological and pathological similarity with human being, as well as the establishment of homologous-recombination-based gene targeting in mouse embryo stem cell, knockout mouse model has taken the center of stage for biomedical investigation for about two decades. As comparing with mouse, rat is more suitable for modeling many human diseases and for investigation in nutriology, toxicology and behavior. However, due to the late development and sophistication in culture of rat embryo stem cell, genetical manipulation in rat has largely lagged behind that of mouse. Even with respect to mouse, because of the intrinsic complexity of ES cell-based gene targeting, genarating of knockout mice is still a skill-demanding, time-consuming and cost-intensive process, which can not properly satisfied the increasing demanding of biomedical research.With recent discovery of the pathological mechanism of phytopathogenic bacteria of the genus Xanthomonas, and the revelation of the adaptive inmune system that protects bacteria and archea from the invasion of phage and plasmid, TALEN (transcription activator-like effector nuclease) and artifical CRISPR/Cas system have increasingly been regarded as prospective tools for genome editing.To investigate the possibility and the efficiency of TALENs to generate gene mutant mice through direct microinjection of TALEN mRNA into zygotes, we first selected 10 target mouse genes from eight different chromosomes. Mutant F0 mice were obtained for each gene with a mutation rate ranged from 13 to 67% and an average of ～40% of total newborns, no significant differences of efficiency were observed between C57BL/6 and FVB/N genetic background. Furthermore, highly efficient germ-line transmission was obtained, as all the FO founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype asdb/db mouse.With directed microinjection of in vitro transcribed RNA for Cas9 and gRNA into zygotes of mouse and rat respectively, mutant mice were obtained with high efficiency with no significant differences of efficiency between C57BL/6 and FVB/N genetic background. By co-injection of Cas9/gRNA against two adjacent sites of Uhrf2 gene into mouse zygotes, long deletion of interval DNA were achieved. By co-injection of Cas9/gRNA against two genes, Mc4r and Mc3r, into rat zygotes, FO rat with double mutant genes were obtained. one of mutant rat that bearing two frame-shifting mutation for Mc4r gene developed obesity-related phenotype that consists with that of ENU-induced Mc4r mutation. Sequencing analysis of potential off-target sites for Th and Rheb genes show no sign of off-target, which indicated reliable specificity for CRISPR/Cas induced genome modification in mice. Efficient germline transmission were achieved for both mutant mouse and rat that were tested. In conclusion, our study demonstrates that TALEN and CRISPR are powerful tools for fast genarating knockout mice and rat with low cost, which can greatly facilitated using mice and rat in biomedical research.