CRISPR/Cas9-mediated GJA8 knockout in rabbits recapitulates human congenital cataracts

Abstract

Cataracts are the leading cause of vision loss in the world, although surgical treatment can restore vision in cataract patients. Until now, there have been no adequate animal models for in vivo studies of artificial lens safety and drug interactions. Genetic studies have demonstrated that GJA8 is involved in maintaining lens opacity and proper lens development. In this study, a cataract model with GJA8 gene knockout was developed via co-injection of Cas9/sgRNA mRNA into rabbit zygotes. Our results showed that gene mutation efficiency in the GJA8 locus reached 98.7% in embryos and 100% in pups, demonstrating that the Cas9/sgRNA system is a highly efficient tool for gene editing in rabbits. In agreement with other studies, our genetic and histology results showed that impaired GJA8 function caused microphthalmia, small lens size and cataracts. In summary, our novel rabbit model of cataracts will be an important drug-screening tool for cataract prevention and treatment.

Figure 1

CRISPR/Cas9-mediated gene targeting of GJA8 in zygotes (A) Schematic diagram of sgRNA targeting the GJA8 gene loci. The yellow rectangle represents the protein coding region of GJA8. Two sgRNAs sequences, sgRNA1 and sgRNA2, are highlighted in red. Protospacer adjacent moti (PAM) sequences are presented in green. Primers F and R were used for mutation detection in embryos and pups. (B) T-cloning and Sanger sequencing of the modified GJA8 alleles in blastocysts. Wild type sequence is shown at the top of the targeting sequence. Insertions are highlighted in blue. E: embryos; WT: wild type; deletion “−”; insertion: “+”. (C) T7E1 cleavage assay for mutation detection in embryos. Gel images have been cropped. Original images are included in “Authors’ original file for Fig. 1C”. Black arrow indicates the WT allele (367 bp). M, DL2000; E1–E9 represents different blastocysts used in this study.

Figure 2. Creation of GJA8 knockout rabbits via zygote injection.

(A) T-cloning and Sanger sequencing in 11 pups with modified GJA8 gene (F0-1-F0-11). Also in “Authors’ original file for Fig. 2A”. sgRNAs sequences are highlighted in red, PAM sequences in green and insertions in blue. WT: wild type; deletion “−”; insertion: “+”. Y: cataract; N: normal. (B) T7E1 cleavage assay for mutation detection in F0 generation pups. Black arrow indicated WT allele (367 bp). M: DL2000; F0-1-F0-11 represented the F0 generation pups used in this study. Gel images have been cropped. Original images are included “Authors’ original file for Fig. 2B”. (C) Western blots of the GJA8 gene knockout rabbit lenses. Equal amounts of protein were loaded, and the β-actin was used as an internal control. (D) Photographs of mutant founder rabbits. Heterozygous GJA8 mutant rabbits (F0-3 and F0-5) with cataracts, Other F0 eyes with cataracts were included in Fig. S1B. (E) H&E staining of WT and GJA8 mutant rabbit lenses. The arrows indicate lenses with cataracts. Scale bar, 50 μm.

Figure 3

Off target detection in the F0 generation of GJA8 knockout rabbits (A) Chromatogram sequence analysis of seven potential off-target sites (POTS) for sgRNA1 using PCR products in founders. 20 bp of the POTS and the PAM are represented in shadow. (B) The chromatogram sequence analysis of seven POTS for sgRNA2 using PCR products in founder rabbits. The 20 bp of the POTS and the PAM are represented in shadow. (C) T7E1 cleavage analysis of POTS for sgRNA1. M, DL2000; 1–7 represent the seven POTS. (D) T7E1 cleavage analysis of POTS for sgRNA2. M, DL2000; 1–7 represent the seven POTS. Gel images have been cropped. Original images are included in “Authors’ original file for Fig. 3C,D”.

Figure 4. Heritability of the GJA8 gene knockout rabbits.

T-cloning and Sanger sequencing analysis of GJA8 knockout rabbit pups. (A) F0-7 crossed with F0-4. (B) F0-5 crossed with F0-8. (C) F0-7 crossed with F0-9. sgRNA sequences are highlighted in red, PAM sequences in green and insertions in blue. WT: wild type; deletions “−”, insertion: “+”. Y: cataract; N: normal.

Figure 5. Phenotype identification of the F1 generation GJA8 knockout rabbits.

(A) T7E1 cleavage assay for mutation detection in pups. M, DL2000; F1-1-F1-24 represent the offspring used in this study. Gel images have been cropped. Original images have been included in “Authors’ original file for Fig. 5A”. Black arrow indicates the WT allele (367 bp). (B) Western blotting from the lens of the GJA8 gene knockout rabbit. Equal amounts of protein were used and β-actin was the internal control. (C) Computer modeling of GJA8 3 D structure and impact of the GJA8 mono-allelic and bi-allelic mutants at the target loci. WT: structure of non-mutant GJA8 gene; F1-4 (WT/-52): GJA8 gene with mono-allelic mutation; F1-1 (−52/−57): GJA8 gene with bi-allelic mutation. (D) Phenotypic comparison of eyes and lens between wild type, bi-allelic and mono-allelic mutant (WT, F1-1 and F1-4) F1 generation rabbits at the age of 13 days old. (E) Photographs of a GJA8 mutant rabbit lens with cataract at the age of 13 days old, WT: wild type; F1-23 (WT/−52), F1-9 (WT/−79). (F) Histology of the GJA8 mutant eyes. Histology data from 3 days old wild type and GJA8 mono-allelic mutant rabbits. (G) Photographs of a GJA8 mutant rabbit lens with cataract at of 3 months old.

Figure 6. Disrupted gap junction in GJA8 KO lens fibers.

(A) Immuolabeling of GJA8 (red) in lens cortical fibers of paraffin sections from WT and GJA8 (+/−) rabbits. Scale bar, 10 μm. (B) Thin-section of intercellular gap junction in the lens cortical fibers from WT and GJA8 (+/−) rabbits. Gap junctions are indicated by yellow arrows. Scale bar, 200 nm.