Efficient dual sgRNA-directed large gene deletion in rabbit with CRISPR/Cas9 system

Abstract

The CRISPR RNA-guided Cas9 nuclease gene-targeting system has been extensively used to edit the genome of several organisms. However, most mutations reported to date have been are indels, resulting in multiple mutations and numerous alleles in targeted genes. In the present study, a large deletion of 105 kb in the TYR (tyrosinase) gene was generated in rabbit via a dual sgRNA-directed CRISPR/Cas9 system. The typical symptoms of albinism accompanied significantly decreased expression of TYR in the TYR knockout rabbits. Furthermore, the same genotype and albinism phenotype were found in the F1 generation, suggesting that large-fragment deletions can be efficiently transmitted to the germline and stably inherited in offspring. Taken together, our data demonstrate that mono and biallelic large deletions can be achieved using the dual sgRNA-directed CRISPR/Cas9 system. This system produces no mosaic mutations or off-target effects, making it an efficient tool for large-fragment deletions in rabbit and other organisms.

Keywords: Albinism; CRISPR/Cas9; Rabbit; Tyrosinase.

Fig. 1

Genome editing of TYR gene via the Cas9/gRNA system. a Schematic diagram of the four sgRNA target sites at the tyrosinase (TYR) gene locus. The CDS region is indicated by blue rectangles. SgRNA target sites are indicated by black arrow. SgRNA1and sgRNA2 are located upstream of the TYR locus. SgRNA3 and sgRNA4 are located in the 3′ UTR. 1/2/3 F and 1/2/3 R represent the PCR primer pairs used for mutation detection. The scale bar represents 10 kb. b Constructs and schematic illustration of the Cas9/gRNA system used in this study. c Target sequence of the four sgRNA used in this study

Fig. 2

Dual sgRNA-directed large deletion of TYR in zygotes. a Determination of 105-kb deletions of TYR gene in rabbit embryos by PCR. A clear band demonstrates the large-fragment deletion of TYR in #5 embryos; primers 1F and 1R were used for large-fragment determination. M DNA ladder. b Mutation detection for each sgRNA in zygotes. Primers TYR-2F/2R were used for mutation determination for sgRNA1 and sgRNA2, and TYR-3F/3R were used for sgRNA3 and sgRNA 4. M DNA marker. c T-cloning sequencing of deletion of TYR in #5 blastocyst. PAM sites are underlined and highlighted in red; target sequences are green; deletions (−) and insertions (+) are shown. WT wild-type control. d T-cloning sequencing of the target site for each sgRNA in injected embryos. PAM sites are underlined and highlighted in red; target sequences are green; deletions (−) and insertions (+) are shown. WT wild-type control

Fig. 3

Generation of large deletion of TYR in rabbits by dual sgRNAs. a Phenotype of TYR mutant rabbit; #301 is the TYR biallelic mutant (homozygote), which exhibited the typical albinism phenotype; #204 and #106 are the TYR monoalleic mutation (heterozygote) and the wild-type rabbit. The box shows the light pink-tinted iris and pupil of the #301 rabbit compared with the dark iris of wild-type rabbits. WT wild-type control. b The mutation determination of 105 kb deletions of TYR gene in founder rabbits by PCR. Results showed that three (#106, #204 and #301) of the 17 founders were large deletion of TYR rabbit. Primers used were TYR-F1 and R1 (Table S1). M DNA marker. c T-cloning sequences of mutant alleles in the large deletion of TYR rabbit (#106, #204 and #301). The PAM sites are underlined and highlighted in red; the target sequences are green; deletions (−) and insertions (+) are shown. WT wild-type control. d Chimera analysis of different tissues from #204 by PCR-sequencing. All detected tissues showed the same PCR band. Primers used were F1 and R1 (Table S1). M DNA marker, WT wild-type control. e T-cloning sequences of mutant alleles in different tissues from #204. PAM sites are underlined and highlighted in red; target sequences are green; deletions (−) and insertions (+) are shown. WT wild-type control

Fig. 4

PCR and T-cloning sequencing of the target site for each sgRNA in founder rabbit. a Mutation detection of sgRNA1 and sgRNA2 in founder rabbit by PCR. Primers TYR-2F/2R were used for mutation determination. M DNA marker, WT wild-type control. b Mutation detection of sgRNA3 and sgRNA4 in founder rabbit by PCR. Primers TYR-3F/3R were used for mutation determination. M DNA marker, WT wild-type control. c T-cloning sequencing of the target sites of sgRNA1 and sgRNA2 in founder rabbits. PAM sites are underlined and highlighted in red; target sequences are green; deletions (−) and insertions (+) are shown. WT wild-type control. d T-cloning sequencing of the target sites of sgRNA3 and sgRNA4 in founder rabbits. PAM sites are underlined and highlighted in red; target sequences are green; deletions (−) and insertions (+) are shown. WT wild-type control

Fig. 5

Phenotype identification of TYR mutated rabbits. a Expression of TYR gene was determined by qRT-PCR. WT wild-type control, NW New Zealand white rabbit. The data were analyzed by t tests using Graphpad Prism software. A probability of P < 0.05 was considered statistically significant. *P < 0.05; **P < 0.01; ***P < 0.005. ns not significant, WT wild-type control. b Western blot and gray-scale analysis of the expression of TYR protein. The image was quantified using ImageJ software (NIH) and all the data are expressed as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.005. WT wild-type control. c H&E staining of the irides and skin from the WT and TYR mutated rabbits. The arrows indicate the melanin in the iris and the basal layer of the epidermis. WT wild-type control

Fig. 6

Generation of F1s carrying large deletions of TYR. a Sequence analysis of rabbit containing a large deletion of TYR (#106) and New Zealand white rabbit. SNP is indicated by the blue arrows. Deletions (−) and insertions (+) are shown. WT wild-type allele, NW New Zealand white rabbit. b Picture of the F1 TYR mutant rabbit; four of the F1 rabbits exhibited the typical albinism phenotype; others are wild-type rabbits. c Determination of large deletions of TYR in F1 rabbits by PCR. A clear band demonstrates a large-fragment deletion of TYR is present in #401, #402, #403, and #404. Primers 1F and 1R were used for mutant determination. M DNA ladder. d T-cloning sequence analysis of the F1 rabbits. SNP is indicated by the blue arrows. Deletions (−) and insertions (+) are shown. WT wild-type allele