Single-step generation of rabbits carrying a targeted allele of the tyrosinase gene using CRISPR/Cas9

Abstract

Targeted genome editing of nonrodent mammalian species has provided the potential for highly accurate interventions into gene function in humans and the generation of useful animal models of human diseases. Here we show successful clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas)-mediated gene targeting via circular plasmid injection in rabbits. The rabbit tyrosinase gene (TYR) was effectively disrupted, and we confirmed germline transmission by pronuclear injection of a circular plasmid expressing humanized Cas9 (hCas9) and single-guide RNA. Direct injection into pronuclear stage zygotes was possible following an in vitro validation assay. Neither off-target mutagenesis nor hCas9 transgenesis was detected in any of the genetically targeted pups and embryos examined. Gene targeting with this rapid and simplified strategy will help accelerate the development of translational research using other nonrodent mammalian species.

Fig. 1.

Scheme for CRISPR/Cas9-mediated gene targeting. (A) Scheme for the SSA assay using pCAG EGxxFP and pX330 in HEK293 cells. The transduced hCas9 and sgRNA complex caused DSB in the target sequence of pCAG-EGxxFP by homology-dependent repair via homologous recombination (HR) or caused single-strand annealing (SSA). (B) Schematic representation of sgRNAs targeting tyrosinase (TYR). The sgRNA target sequence and PAM sequence are highlighted in black and red, respectively.

Fig. 2.

Evaluation of sgRNA sequences for disrupting the rabbit tyrosinase gene. (A) The efficiency of DSB-mediated homology-dependent repair was validated by observing EGFP fluorescence at 24 h after transfection (left panels, EGFP signals; right panels, phase contrast microscopy images). (N.C., normal control pX330 without an sgRNA sequence; CR1-CR4, pX330 plasmid with tyrosinase CR1/CR2/CR3/CR4 sequences, respectively). (B) Quantitative representation of the DSB efficiency of each sgRNA sequence. Data are shown as the mean ± standard deviation (SD). Scale bar=100 µm.

Fig. 3.

CRISPR/Cas9-mediated modification of the tyrosinase gene in founder rabbits. (A) Five-day-old founder pups with representative tyrosinase genomic sequences (top panels, wild-type pup; bottom panels, heterozygous pup with a 2-bp deletion). The arrow indicates the indel mutation in this pup. Subcloning and sequencing its PCR fragment confirmed each allele’s identity. (B) Photograph and representative tyrosinase genomic sequences of F1 pups that showed a Dutch-like coat color (closed arrow) and JW-like (white) coat color (open arrows). (C) Photograph of JW-like does that have a white coat color and red eyes.