The CRISPR-Cas system is a powerful tool for generating genetically modified animals; however, targeted knock-in (KI) via homologous recombination remains difficult in zygotes. Here we show efficient gene KI in rats by combining CRISPR-Cas with single-stranded oligodeoxynucleotides (ssODNs). First, a 1-kb ssODN co-injected with guide RNA (gRNA) and Cas9 messenger RNA produce GFP-KI at the rat Thy1 locus. Then, two gRNAs with two 80-bp ssODNs direct efficient integration of a 5.5-kb CAG-GFP vector into the Rosa26 locus via ssODN-mediated end joining. This protocol also achieves KI of a 200-kb BAC containing the human SIRPA locus, concomitantly knocking out the rat Sirpa gene. Finally, three gRNAs and two ssODNs replace 58-kb of the rat Cyp2d cluster with a 6.2-kb human CYP2D6 gene. These ssODN-mediated KI protocols can be applied to any target site with any donor vector without the need to construct homology arms, thus simplifying genome engineering in living organisms.

Embryonic appendicular structures, such as the limb buds and the developing external genitalia, are suitable models with which to analyze the reciprocal interactions of growth factors in the regulation of outgrowth. Although several studies have evaluated the individual functions of different growth factors in appendicular growth, the coordinated function and integration of input from multiple signaling cascades is poorly understood. We demonstrate that a novel signaling cascade governs formation of the embryonic external genitalia [genital tubercle (GT)]. We show that the dosage of Shh signal is tightly associated with subsequent levels of Wnt/-catenin activity and the extent of external genitalia outgrowth. In Shh-null mouse embryos, both expression of Wnt ligands and Wnt/-catenin signaling activity are downregulated. -catenin gain-of-function mutation rescues defective GT outgrowth and Fgf8 expression in Shh-null embryos. These data indicate that Wnt/-catenin signaling in the distal urethral epithelium acts downstream of Shh signaling during GT outgrowth. The current data also suggest that Wnt/-catenin regulates Fgf8 expression via Lef/Tcf binding sites in a 3Ј conserved enhancer. Fgf8 induces phosphorylation of Erk1/2 and cell proliferation in the GT mesenchyme in vitro, yet Fgf4/8 compound-mutant phenotypes indicate dispensable functions of Fgf4/8 and the possibility of redundancy among multiple Fgfs in GT development. Our results provide new insights into the integration of growth factor signaling in the appendicular developmental programs that regulate external genitalia development.

Transcription activator-like effector (TALE) nuclease (TALEN) is a site-specific nuclease, which can be freely designed and easily constructed. Numerous methods of constructing TALENs harboring different TALE scaffolds and repeat variants have recently been reported. However, the functionalities of structurally different TALENs have not yet been compared. Here, we report on the functional differences among several types of TALENs targeting the same loci. Using HEK293T cell-based single-strand annealing and Cel-I nuclease assays, we found that TALENs with periodically-patterned repeat variants harboring non-repeat-variable di-residue (non-RVD) variations (Platinum TALENs) showed higher activities than TALENs without non-RVD variations. Furthermore, the efficiencies of gene disruption mediated by Platinum TALENs in frogs and rats were significantly higher than in previous reports. This study therefore demonstrated an efficient system for the construction of these highly active Platinum TALENs (Platinum Gate system), which could establish a new standard in TALEN engineering.

Engineered endonucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, provide a powerful approach for genome editing in animals. However, the microinjection of endonucleases into embryos requires a high skill level, is time consuming, and may cause damage to embryos. Here, we demonstrate that the electroporation of endonuclease mRNAs into intact embryos can induce editing at targeted loci and efficiently produce knockout rats. It is noteworthy that the electroporation of ZFNs resulted in an embryonic survival rate (91%) and a genome-editing rate (73%) that were more than 2-fold higher than the corresponding rates from conventional microinjection. Electroporation technology provides a simple and effective method to produce knockout animals.

The bacterial CRISPR/Cas system has proven to be an efficient gene-targeting tool in various organisms. Here we employ CRISPR/Cas for accurate and efficient genome editing in rats. The synthetic chimeric guide RNAs (gRNAs) discriminate a single-nucleotide polymorphism (SNP) difference in rat embryonic fibroblasts, allowing allele-specific genome editing of the dominant phenotype in (F344 × DA)F1 hybrid embryos. Interestingly, the targeted allele, initially assessed by the allele-specific gRNA, is repaired by an interallelic gene conversion between homologous chromosomes. Using single-stranded oligodeoxynucleotides, we recover three recessive phenotypes: the albino phenotype by SNP exchange; the non-agouti phenotype by integration of a 19-bp DNA fragment; and the hooded phenotype by eliminating a 7,098-bp insertional DNA fragment, evolutionary-derived from an endogenous retrovirus. Successful in vivo application of the CRISPR/Cas system confirms its importance as a genetic engineering tool for creating animal models of human diseases and its potential use in gene therapy.