CRISPR/Cas9-mediated correction of MITF homozygous point mutation in a Waardenburg syndrome 2A pig model

Abstract

Gene therapy for curing congenital human diseases is promising, but the feasibility and safety need to be further evaluated. In this study, based on a pig model that carries the c.740T>C (L247S) mutation in MITF with an inheritance pattern and clinical pathology that mimics Waardenburg syndrome 2A (WS2A), we corrected the point mutation by the CRISPR-Cas9 system in the mutant fibroblast cells using single-stranded oligodeoxynucleotide (ssODN) and long donor plasmid DNA as the repair template. By using long donor DNA, precise correction of this point mutation was achieved. The corrected cells were then used as the donor cell for somatic cell nuclear transfer (SCNT) to produce piglets, which exhibited a successfully rescued phenotype of WS2A, including anophthalmia and hearing loss. Furthermore, engineered base editors (BEs) were exploited to make the correction in mutant porcine fibroblast cells and early embryos. The correction efficiency was greatly improved, whereas substantial off-targeting mutations were detected, raising a safety concern for their potential applications in gene therapy. Thus, we explored the possibility of precise correction of WS2A-causing gene mutation by the CRISPR-Cas9 system in a large-animal model, suggesting great prospects for its future applications in treating human genetic diseases.

Keywords: CRISPR/Cas9; MITF; anophthalmia; base editor; gene correction; hearing loss.

Graphical abstract

Figure 1

CRISPR-Cas9 mediated MITF c.740C > T repair in porcine fibroblast cells using ssODN as the repair template (A) Experimental design for the MITF point-mutation repair in the current study. (B) Genome structure of the porcine MITF gene, the sequences of the sgRNAs targeting the c.740 site, and the ssODNs used as HDR templates. The protospacer-adjacent motif (PAM) sequences of the sgRNAs are shown in purple, the intended point mutation is shown in red, and the introduced blocking mutations are shown in blue in the ssODN sequence. (C) Experimental procedure for screening single-cell colonies. (D) Genotyping of single-cell colonies by PCR and DraI digestion that originated from MITF^L247S/L247S fibroblasts. The arrows indicate potentially corrected colonies. (E) Sanger sequencing of the colonies cut by DraI. The T base intended to be repaired is shown in red, the blocking mutations in donor DNA are shown in orange, the deleted bases are indicated by colons, and the inserted bases are shown by lowercase red text. (F) Sequencing diagram of the PCR product from colony 27 (#27) and the MITF^L247S/L247S fibroblast cells (Homo). The sgRNA sequences are underlined, the PAM sequences of the sgRNA1 are shown in red rectangles, and the repaired base and the introduced blocking mutation are indicated by black and red arrows, respectively. (G) Genotyping of single-cell colonies by PCR and DraI digestion originate from MITF^L247S/+ fibroblasts. The arrows indicate the potentially corrected colonies. (H) Sanger sequencing of the colonies cut by DraI. The T base intended to be repaired is shown in red, the blocking mutations in donor DNA are shown in orange, the deleted bases are indicated by colons, and the inserted bases are shown by lowercase red text. (I) Sequencing diagram of the PCR product from colony 3, 5, and 13 and the MITF^L247S/+ fibroblast cells (Hetero). The PAM sequences of the sgRNA1 are shown in red rectangle, and the repaired base and the introduced blocking mutation are indicated by black and red arrows, respectively.

Figure 2

Generation of MITF c.740C>T repaired pigs and phenotype analysis (A) Photographs of pigs with different genotypes are shown. Homo represents the MITF^L247S/L247S pig, Hetero represents the MITF^L247S/+ pig, WT represents the wild-type (MITF^+/+) pig, and Repaired represents the MITF c.740C>T repaired pig. (B) A short band (indicated by arrows) was detected in mRNA from colony 27-derived pigs using primers MITF-M2F and MITF-M2R. M represents the DNA marker. (C) RT-PCR and DraI digestion were used to genotype mRNAs obtained from the ear tissues of colony 27-derived pigs. (D) Representative sequence of the mutant allele in MITF mRNA. The repaired allele is shown above, which included an introducing mutation shown in green and a repaired base shown in purple bold. The deleted bases are indicated by colons, and the inserted bases are shown by blue uppercase text. (E) H&E staining of the eye sections in different groups of pigs. Scale bar, 100 μm. (F) Comparison of the ABR thresholds in piglets with different genotypes at 2 weeks of age; the numbers of pigs for the WT, hetero, homo, and repaired groups are 4, 4, 3 and 3, respectively. The ABR thresholds were recorded in both ears and are presented as mean ± SEM. ∗∗p < 0.01. (G and H) In different groups of pigs, scotopic and photopic ERGs were performed and are shown as average b-wave amplitudes. Analysis included both eyes of 4 WT, 3 hetero, and 2 MITF-repaired pigs. Data are presented as mean ± SEM. Results of the homo group are not shown in (G) and (H) since their eyes were severely hypoplasia (anophthalmia) and cannot be subjected to the ERG experiment. NS, not significant (p > 0.05).

Figure 3

CRISPR-Cas9-mediated MITF point mutation repair in MITF^L247S/L247S porcine fibroblast cells using long donor plasmids as the repair templates (A) Design of different sgRNAs around exon 8 of the porcine MITF gene for the long donor DNA strategy. The PAM sequences of sgRNAs are shown in purple, and the intended point mutation is shown in red. (B) Design of donor DNA and sgRNAs for genome editing. The homology arms are shown in blue and orange lines around the intended T locus, and the different sgRNAs are shown as lightning bolts. The purple lightning bolt represents sgRNA4, the green lightning bolt represents sgRNA3, and the red lightning bolt represents sgRNA1. (C) Analysis of the HDR efficiency for the three different schemes. A representative restriction fragment length polymorphism (RFLP) image of transfected cells is shown on the left, and the Hi-TOM quantitative results of three independent replicates are presented as mean ± SEM (n = 3) on the right. ∗∗p < 0.01. HR, DH, and SH represent HR, DHMEJ, and SHMEJ, respectively. (D) Genotyping of single-cell colonies by PCR and DraI digestion. The arrows indicate the potential repaired colonies. (E) Sanger sequencing of the colonies that were precisely repaired at the intended locus. The biallelically corrected colonies are shown in bold. The T base intended to be repaired is shown in red, the blocking mutations in the donor DNA are shown in orange, and the omitted bases are shown by ellipses. (F and G) Total RNAs were extracted from these colonies and amplified using RT-PCR (F) and then digested with DraI (G). Arrows indicate cleavage. (H) Sequencing diagram of the RT-PCR product of colony HR-10, HR-15, and HR-30 and the MITF^L247S/L247S fibroblast cells (Homo). The intended corrected base and the introduced blocking mutation are indicated by arrows in black and red, respectively. The blue arrow indicates the junction of exon 7 and exon 8 of MITF.

Figure 4

Generation of fully corrected pigs and analysis of their phenotypes (A) Representative photographs of piglets with different genotypes. Homo mutant pig represents the MITF^L247S/L247S pig. (B) Genotyping of genomic DNA obtained from ear tissue of the HR-15 colony-derived piglets by PCR and DraI digestion. Ctrl represent the MITF^L247S/L247S fibroblast cells, and 7601-06, 7801-02, and 7901 represent the repaired pigs. WT1–2 represents two WT pigs. The arrow indicates the cut band with the repaired sequence. (C and D) Retina function analysis in repaired pigs. Scotopic (rod, C) and photopic (cone, D) full-field ERGs were recorded in newborn piglets; representative scotopic and photopic records are shown for the repaired pigs and WT pigs. (E and F) The b-wave amplitudes of scotopic (E) and photopic (F) full-field ERGs were plotted for both eyes of the repaired (n = 3) and WT pigs (n = 3). Data are presented as mean ± SEM. ∗∗p < 0.01. NS, not significant (p > 0.05). Results of the homo group are not shown in (C–F) since their eyes were severely hypoplasia (anophthalmia) and could not be subjected to the ERG experiment. (G) Representative H&E staining image of the retina of newborn piglets with different genotypes. Scale bar, 50 μm. (H–J) Representative images of the ABR test in newborn piglets with different genotypes. (K) Comparison of the ABR threshold in newborn piglets with different genotypes; the numbers of pigs for WT, homozygous mutant, and the repaired group are 3, 2 and 1, respectively. The ABR thresholds were recorded in both ears. Data are presented as mean ± SEM. ∗∗p < 0.01.

Figure 5

Correction of the MITF c.740T>C mutation using BEs (A) RFLP analysis in MITF^L247S/L247S fibroblast cells that were transfected with different BEs as indicated. (B) Hi-TOM was used to evaluate the editing efficiency of the intended correction with different BEs. The sequences with C9 corrected to T or both C9 and C4 mutated to T were counted as corrected events. Data are presented as mean ± SEM (n = 3 from three independent experiments). a, b, and c values with no letter in common are significantly different (p < 0.05). (C) Representative Sanger sequencing diagrams of the corrected colonies compared with the MITF^L247S/L247S cells (Homo). The sgRNA sequences are underlined, and the PAM sequences are shown in red rectangles. The edited bases are indicated by arrows, the black arrows indicate the intended mutations (C9), and the red arrows indicate the C4 mutations in the editing window. (D) Schematic of microinjection in MITF^L247S/L247S and MITF^L247S/+ fibroblast-derived embryos. (E) Summary of the editing efficiency in heterozygous (hetero) and homozygous (homo) embryos by microinjection of sgRNA and hA3A-eBE-Y130F mRNA. Sequences with C9 corrected to T/T and C3 unedited were counted as intended mutants.

Figure 6

Analysis of the off-targeting mutations identified by WGS (A) The chromosomal distribution of all SNVs and indels throughout the porcine genome. (B and C) Number of all SNVs (B) and indels (C) identified in the three samples. (D) Overlap of all of the SNVs and indels between the three samples. (E) Frequencies of different types of all SNVs detected in the corrected colonies and pig. (F) Overlap of all the SNVs and indels detected by WGS with predicted off-target sites by Cas-OFFinder and CRISPOR. (G–I) The distribution analysis of all SNVs (G), C>T/G>A SNVs (H), and all indels (I) in the given regions.