Functional validation of the albinism-associated tyrosinase T373K SNP by CRISPR/Cas9-mediated homology-directed repair (HDR) in rabbits

Abstract

Background: Oculocutaneous albinism (OCA) is a group of autosomal recessive disorders characterized by reduced melanin that are caused by mutations in the gene encoding tyrosinase (TYR), which is the rate-limiting enzyme in the production of the pigment melanin. Many studies or meta-analyses have suggested an association between the TYR T373K SNP and OCA1, but there is limited biochemical and genetic evidence to support this association.

Methods: We overexpressed TYR-WT and TYR-T373K mutants on HK293T cells and tested the changes of melanin production and tyrosinase activity. Then we generated TYR-K373T knock-in (KI) rabbits by microinjection of ssDNA and synthesized RNAs targeting C1118A using CRISPR/Cas9-HDR to observe the formation of melanin.

Findings: We demonstrated that the T373K mutation in TYR can reduce tyrosinase activity, leading to an absence of melanin synthesis at the cell-level. The gene-edited TYR-K373T rabbits exhibited rescued melanin production in hair follicles and irises, as inferred from the evident decrease in pigmentation in TYR-T373K rabbits, thus providing functional validation of the albinism-associated T373K SNP at the animal level.

Interpretation: Our study provides the first animal-level functional validation of the albinism-associated TYR K373T SNP in rabbits, and these results will facilitate gene therapy of OCA1 in pre-clinical settings in the future. FUND: The National Key Research and Development Program of China Stem Cell and Translational Research, the Strategic Priority Research Program of the Chinese Academy of Sciences, the Guangdong Province Science and Technology Plan Project, and the Program for JLU Science and Technology Innovative Research Team.

Keywords: CRISPR/Cas9; OCA; Point mutation; Rabbit; T373K; Tyrosinase.

Fig. 1

Reduced melanin synthesis in TYR-T373K mutant cells. (A) Schematic representation of the sequence of human tyrosinase CYS-rich, regions rich in conserved cysteine residues; CuA and CuB, proposed binding sites for the two copper atoms based on sequence comparisons with other copper-binding proteins. (B) Overexpression of the WT-TYR and TYR-T373K mutant genes in HEK293T cells. (C) The colours of the transfected cell pellets were compared between the WT-TYR- and TYR-T373K-transfected cells. (D) mRNA half-lives were compared between WT-TYR and TYR-T373K. (E) Gene mRNA expression was compared between WT-TYR and TYR-T373K. (F) Melanin content was compared between the WT-TYR- and TYR-T373K-transfected cells. (G) Tyrosinase activity was compared between WT-TYR and TYR-T373K. Mock, negative control. ***, p < .001.

Fig. 2

Editing of the TYR gene via the Cas9/gRNA system in zygotes. (A) Schematic diagram depicting the TYR-K373T SNP in albino rabbits and mice. The K373T SNP is shown in the red box and is indicated by the arrows to be present in rabbits but not mice. (B) Diagram of the sgRNA and donor ssODNs used in this study. The sgRNA is marked in green, and the point mutation is shown in red. The base C was replaced with T, producing a single precision point mutation, resulting in K373T (AAG-ACG). Two synonymous mutations near the protospacer adjacent motif (PAM) were introduced in the PAM sequence (red). The long sequence shown at the bottom is that of an ssODN with the desired mutations, synonymous mutations and homologous arms (45 bp to the left and right). (C) Genome editing of the TYR gene in blastocysts by PCR. B1–B14 represent the different blastocysts used in this study, M, DNA marker D2000. (D) Editing of the TYR gene in KI blastocysts was verified by T-cloning and Sanger sequencing. The WT sequence is shown above the target sequence. The sgRNA sequence is marked in green, and the PAM sequences are marked in red and underlined. WT, wild type; deletion, “−”; insertion, “+”.

Fig. 3

Generation of TYR-K373T mutant rabbits by CRISPR/Cas9. (A, B) Mutation detection in founder rabbits by PCR and the T7E1 assay. M, DNA marker D2000. 101–403 represent the different pups used in this study. (C, D) Mutation detection in founder rabbits by T-cloning and Sanger sequencing. The PAM sites are underlined and highlighted in red; the target sequences are shown in green; deletions (−) and insertions (+) are indicated. WT, wild type. (E) GUIDE-seq results for sgRNA-targeted genomic sites in TYR-K373T mutant rabbits. Chromatogram sequence analysis of sgRNA-targeted genomic sites. The red arrows represent the mutation sites, and the numbers represent the sgRNA base position from the 5′ end. The percentage of sequencing reads harbouring the mutation relative to the total reads is shown. The numbers represent the sgRNA base position from the 5′ end. Values and error bars reflect the means and SDs of n = 3 biologically independent samples.

Fig. 4

Increased melanin production in TYR-K373T rabbits. (A) Black hair and eyes in TYR-K373T rabbits. (B) Pathological analysis of the skin and irises from WT-T373K and TYR-K373T rabbits. (C) Gene expression levels of TYR were determined by qRT-PCR. (D) Tyrosinase protein levels were determined by western blotting. (E) Grey-scale analysis of the tyrosinase protein by ImageJ software. All data are expressed as the mean ± SEM. K373T-Hom, homozygous mutation of K373T; K373T-Chi, chimaeric mutation of K373T; WT-T373K, New Zealand white rabbit with a natural T373K mutation.

Fig. 5

Heritability of the TYR-K373T mutation in rabbits. (A) A male K373T-Het rabbit (#307) was mated with female wild-type white rabbits (WT-T373K), and F1 pups with black skin colour were obtained. (B) A male K373T-Chi rabbit (#106) was mated with female WT-T373K white rabbits, and F1 pups with black skin colour were obtained. T-cloning sequence analysis of the F1 rabbits. The PAM sites are highlighted in blue; the target sequences are shown in green; the point mutation sequences are shown in red. Deletions (−) and insertions (+) are indicated. (C) HE staining of the skin and irises from WT-T373K and K373T- F1 rabbits. K373T-Het, heterozygous mutation of K373T; K373T-Chi, chimaeric mutation of K373T; WT-T373K, New Zealand white rabbit with a natural T373 K mutation.