Effective gene targeting in rabbits using RNA-guided Cas9 nucleases.

Dear Editor, Recently, zinc finger nuclease, transcription activator-like effector nuclease, and RNA-guided Cas9 endonuclease (Cas9) have emerged as powerful means for genome editing (Conklin, 2013; Gaj et al., 2013). These nucleases are efficient in generating double-strand breaks in the genome that can be repaired by error-prone nonhomologous end joining leading to a functional knockout (KO) of the targeted gene or used to integrate a DNA sequence at a specific locus through homologous recombination. Although the Cas9 system has been shown highly efficient in generating genetically engineered mice and rats (Li et al., 2013a, b; Wang et al., 2013), its feasibility in the rabbits still needs be determined. Here we report the use of Cas9 system to effectively generate targeted mutations in rabbit embryos and the production of KO rabbits. The rabbit is a classic model animal species. It is useful for the study of many human diseases such as atherosclerosis, cystic fibrosis, and acquired immunodeficiency syndrome. However, production of gene targeted transgenic (GTT) rabbits has been an extreme challenge. This is mainly due to the lack of germline transmitting embryonic stem cells and the very low efficiency of somatic cell nuclear transfer in rabbits (Chesne et al., 2002). In the present work, we used the Cas9 system to target the rabbit genome. Because embryo transfer work in large animal species is costly, we first established an in vitro system to test the efficacy of the RNA-guided Cas9 nucleases. Individual single guide RNAs (sgRNAs) were designed (Figure 1A and Supplementary Table S1) to target nine rabbit genes: apolipoprotein E (APOE), cluster of differentiation 36 (CD36), cystic fibrosis transmembrane conductance regulator, low-density lipoprotein receptor (LDLR), apolipoprotein CIII, scavenger receptor class B, member 1 (SCARB1), leptin, leptin receptor, and ryanodine receptor 2 (RyR2). For each gene, RNA mixture of Cas9 constructs (150 ng/ml Cas9 mRNA plus 6 ng/ml sgRNA) was microinjected into cytoplasm of pronuclear stage rabbit embryos (n 1⁄4 290). We chose to use the concentration at 6 ng/ml for sgRNA because higher concentrations (12, 18, or 24 ng/ml) did not significantly improve the mutation rates (data not shown), and we speculate that higher quantity of sgRNA may increase the frequency of off-target events. Embryos were cultured in vitro, collected at blastocyst (BL) stage, and subjected to single embryo PCR and sequencing (n 1⁄4 116) to identify mutations in the corresponding target locus. All nine sgRNAs generated mutations on their corresponding targeting loci with efficiencies ranging from 10% to nearly 100% (Figure 1D). High percentage (four out of nine) of the sgRNAs resulted in mutation rates higher than 50%; interestingly, bi-allelic mutations were also identified in these four, but not in those where mutation rates were 50% or lower (Figure 1D). After validating the in vitro gene targeting capacity of these Cas9 constructs, we continued to use four of them (APOE, CD36, LDLR, and RYR2) to produce KO rabbits. CD36, LDLR, and APOE KO rabbits are useful to study lipid metabolisms and atherosclerosis. The fourth line (RyR2 KO) will be used as a model to study heart arrhythmia. This is in recognition of the increasing demand for nonmurine models for cardiovascular diseases in the research community. Cardiac physiology of rabbit better mimics that of human in a number of key areas than mouse does (Fan and Watanabe, 2003). For example, cholesteryl ester transfer protein, which plays a central role in the atherosclerotic process, is abundant in both human and rabbit plasma but absent in the mouse. Like humans, rabbits are very susceptible to diet-induced atherosclerosis, whereas wild-type (WT) mice do not develop atherosclerosis naturally. A total of 301 embryos were injected with one of the four Cas9 constructs and transferred to 10 pseudo-pregnant recipient rabbits (20–35 embryos per recipient). After 1 month gestation, nine (90%) recipients gave birth to 68 live kits (7.6/L), out of which 38 were identified as positive KO after initial T7 endonuclease assay and final confirmation by PCR sequencing (Figure 1B, C, and E). The term rate calculated as total term kits/total embryos is 22.6% (68/301). The KO rate calculated as total KO kits/total term is 55.9% (38/68). Consistent with the prediction based on the in vitro results, three (i.e. APOE, CD36, and RyR2) out of the four Cas9 constructs resulted in higher than 50% mutation rates and bi-allelic mutations. It remains to be tested whether mutations in these founder animals will faithfully transmit to thenextgeneration. Inagreement withprevious reports,D15 mutant alleles were repeatedly discovered in six out of nine LDLR KO founder rabbits (Figure 1C, left lower panel), likely caused by microhomologymediated end joining (Wang et al., 2013). One main concern with the Cas9 system for gene targeting is the off-target effects (Fu et al., 2013; Hsu et al., 2013). Recently, Hsu et al. (2013) examined Cas9-induced off-target mutation events in human 293T and 293FT cells. They found that sgRNA can tolerate as much as 4 nt changes in the seed sequence, and that the change of the protospacer adaptor motif (PAM) sequence from NGG to NAG does not totally abolish the targeting capacity. Their work suggested that there may be up to hundreds of potential off-target loci in a mammalian genome for one particular Cas9 seed sequence. In the present work, we examined off-target effects in all the KO founders (LDLR, RYR2, CD36, and APOE), following similar strategies described by Wang et al. (2013) in their mouse work. We used BLASTn to identify exact match to the 15 nt doi:10.1093/jmcb/mjt047 Journal of Molecular Cell Biology (2014), 6, 97–99 | 97