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, 17, 156-173
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Genome Editing in Patient iPSCs Corrects the Most Prevalent USH2A Mutations and Reveals Intriguing Mutant mRNA Expression Profiles

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Genome Editing in Patient iPSCs Corrects the Most Prevalent USH2A Mutations and Reveals Intriguing Mutant mRNA Expression Profiles

Carla Sanjurjo-Soriano et al. Mol Ther Methods Clin Dev.

Abstract

Inherited retinal dystrophies (IRDs) are characterized by progressive photoreceptor degeneration and vision loss. Usher syndrome (USH) is a syndromic IRD characterized by retinitis pigmentosa (RP) and hearing loss. USH is clinically and genetically heterogeneous, and the most prevalent causative gene is USH2A. USH2A mutations also account for a large number of isolated autosomal recessive RP (arRP) cases. This high prevalence is due to two recurrent USH2A mutations, c.2276G>T and c.2299delG. Due to the large size of the USH2A cDNA, gene augmentation therapy is inaccessible. However, CRISPR/Cas9-mediated genome editing is a viable alternative. We used enhanced specificity Cas9 of Streptococcus pyogenes (eSpCas9) to successfully achieve seamless correction of the two most prevalent USH2A mutations in induced pluripotent stem cells (iPSCs) of patients with USH or arRP. Our results highlight features that promote high target efficacy and specificity of eSpCas9. Consistently, we did not identify any off-target mutagenesis in the corrected iPSCs, which also retained pluripotency and genetic stability. Furthermore, analysis of USH2A expression unexpectedly identified aberrant mRNA levels associated with the c.2276G>T and c.2299delG mutations that were reverted following correction. Taken together, our efficient CRISPR/Cas9-mediated strategy for USH2A mutation correction brings hope for a potential treatment for USH and arRP patients.

Figures

Figure 1
Figure 1
Design and Selection of the Optimal sgRNA and ssODN (A) Sequence of exon 13 of USH2A. Red arrowheads indicate the c.2276G>T and c.2299delG mutations. The four different sgRNAs (sgRNA-1 to sgRNA-4) are underlined. The green boxes correspond to the 3′ PAM sequences adjacent to each sgRNA. (B) Representative gel image of a T7E1 assay in HEK293 cells for each sgRNA. Untreated DNA from the same HEK293 cells was used as a negative control (C−). Samples not treated with T7E1 are indicated by a minus sign. Samples treated with T7E1 are indicated by a plus sign. (C) Representative gel image of a T7E1 assay in HEK293 cells for each sgRNA. The absence or presence of the mismatched G in the sgRNA sequence is indicated as “−G” or “+G,” respectively. Untreated DNA from the same HEK293 cells was used as a negative control (C−). Samples not treated with T7E1 are indicated by a minus sign. Samples treated with T7E1 are indicated by a plus sign. (D) Diagram showing the design of the exogenous repair templates ssODN-1 (upper panel) and ssODN-2 (lower panel). The position of the PAM sequences is shown in green, and green arrowheads indicate the PAM silent mutations introduced into the ssODNs. The restriction enzyme digestion sites for NcoI and MscI in ssODN-1 and ssODN-2, respectively, are boxed in blue. In ssODN-1, a red arrowhead indicates the guanine substitution to correct the c.2276G>T mutation. In ssODN-2, a red arrowhead indicates the inclusion of the cytosine (antisense allele) to correct the c.2299delG mutation.
Figure 2
Figure 2
Gene Correction of the c.2299delG Mutation in the USH2A-USH-iPSC Cell Line (A) MscI restriction enzyme digestion of DNA from the surviving USH2A-USH-iPSC clones B1F11, B3B8, B3B1, B2H4, and B2A3. DNA from the parental USH2A-USH-iPSC line was used as a negative control (C−). (B) Electropherograms showing the Sanger sequencing results of CRISPR/Cas9-mediated genome correction of the c.2299delG mutation: (left panel) heterozygous correction; (right panel) homozygous correction (boxed in red). The incorporated PAM silent mutation is boxed in green. (C) Sequence analysis of allele 1 (A1) and allele 2 (A2) from the CRISPR/Cas9-edited region after subcloning. The red arrowhead indicates the c.2299delG mutation. PAM sequences are boxed by green. The green arrowhead shows the PAM silent mutation present in ssODN-2. (D) qPCR analysis of copy number variation (CNV) of USH2A in the corrected clones compared with that of the parental USH2A-USH-iPSC line (C−). The data were normalized to the housekeeping genes TERT (left panel) and TRMT10C (right panel).
Figure 3
Figure 3
Cas9 Allele-Specific Cleavage In trans to c.2276G>T in the USH2A-RP-iPSC Cell Line (A) Representative NcoI restriction enzyme digestion of 14 of the surviving USH2A-RP-iPSC clones. DNA from the parental USH2A-RP-iPSC was used as a negative control (C−). (B) Representative sequence analysis of allele 1 (A1) and allele 2 (A2) in the 14 clones shown in (A). Highlighted in red are the positions of the mutations c.2276G>T and c.2299delG. Green boxes represent PAM sequences. The green arrowhead shows the position of the PAM silent mutation present in ssODN-1. Black dashes correspond to the Cas9-induced INDELs, which were determined by comparison with the wild-type USH2A reference sequence. (C) Sequence analysis of A1 and A2 from the parental USH2A-RP-iPSC cell line after subcloning. Red arrowheads indicate the c.2276G>T and c.2299delG mutations. Green boxes represent the PAM sequences. A blue arrowhead indicates the SNP c.2256T>C (rs111033281) present in A1.
Figure 4
Figure 4
Gene Correction of the c.2276G>T Mutation in the USH2A-RP-iPSC Cell Line (A) Sequence design for sgRNA-1S that targets allele 1 (A1) carrying the SNP c.2256T>C (blue arrowhead), in comparison with sgRNA-1, which targets allele 2 (A2). Green boxes represent the PAM sequences. Red arrowheads indicate the mutations c.2276G>T and c.2299delG in A1 and A2, respectively. (B) Schematic representation of the proportion of CRISPR/Cas9-induced modifications in A1 (black) and in A1 and A2 (gray). (C) Sequence analysis of A1 and A2 in the surviving USH2A-RP-iPSC corrected clones. Green boxes represent PAM sequences, and the green arrowhead indicates the PAM silent mutation present in ssODN-1. The blue arrowhead indicates the SNP present in A1. Red arrowheads indicate the mutations c.2276G>T on A1 and c.2299delG on A2. Black dashes correspond to the Cas9-induced INDELs, which were determined by comparison with the wild-type USH2A reference sequence. (D) Electropherogram showing the results from Sanger sequencing of CRISPR/Cas9-mediated genome correction of the c.2276G>T mutation in the USH2A-RP-iPSC MS3F7 clone. The corrected c.2276G nucleotide is boxed in red. A dashed red box indicates the uncorrected trans allelic c.2299delG mutation. A blue box indicates the SNP, and a green box indicates the PAM silent mutation.
Figure 5
Figure 5
Whole-Exome Sequencing of Parental and Corrected iPSC Lines (A and D) Exonic predicted off-target sites using the MIT portal for sgRNA-2 (A) and sgRNA-1 (D, upper panel) or the CRISPOR website for sgRNA-1S (D, lower panel) are listed. The number of mismatched nucleotides (MM) is shown and represented by green letters. (B and E) Circos plots indicating the variants identified by WES in the parental and corrected USH2A-USH-iPSC (B) and USH2A-RP-iPSC (E) clones. External layer represents the variants (SNVs + INDELs) shared between the parental and corrected iPSCs. The middle layer displays the single-nucleotide variants (SNVs) exclusively identified in the corrected iPSCs. The internal layer shows the INDELs exclusively identified in the corrected iPSCs. The middle and internal layers exclude variants listed in dbSNP151 but include all variants identified prior to manual inspection. Red bars: homozygous variants; blue bars: heterozygous variants. (C and F) Variants retained after filtering in the corrected USH2A-USH-iPSC clone B3B1 (C) and USH2A-RP-iPSC clone MS3F7 (F). Sequences flanking the variants (in red) in comparison with the corresponding sgRNA target sequence; PAM is boxed in green.
Figure 6
Figure 6
Genome Stability and Pluripotency of Corrected iPSC Lines (A and I) Phase-contrast images of USH2A-USH-iPSC clone B3B1 (A) and USH2A-RP-iPSC clone MS3F7 (I). (B and J) Genomic stability of USH2A-USH-iPSC B3B1 (B) and USH2A-RP-iPSC MS3F7 (J) as determined by a digital qPCR analysis of the most commonly rearranged regions reported in iPSCs. The copy number for each chromosomal position is shown with colored dots. (C–E and K–M) Pluripotency of USH2A-USH-iPSC clone B3B1 and USH2A-RP-iPSC clone MS3F7 as determined by immunostaining of the markers OCT3/4 (C and K), SOX2 (D and L), and NANOG (E and M), respectively. Scale bars, 50 μM. (F-H and N-P) Differentiation capacity of USH2A-USH-iPSC clone B3B1 and USH2A-RP-iPSC clone MS3F7 as determined by immunostaining of the germ layer markers GFAP (ectoderm; F and N), SMA (mesoderm; G and O), and AFP (endoderm; H and P), respectively. Scale bars, 20 μM.
Figure 7
Figure 7
USH2A mRNA Expression Levels in Parental and Corrected iPSCs (A and B) USH2A mRNA expression levels in exon 39 (A) and exon 13 (B) for USH2A-USH-iPSC and the corrected USH2A-USH-iPSC clone B3B1. (C and D) USH2A mRNA expression levels in exon 39 (C) and exon 13 (D) for USH2A-RP-iPSC and the corrected USH2A-RP-iPSC clone MS3F7. Wild-type iPSC (WT) was used as a control, and its relative expression was set at 1. The allelic (A1 and A2) mutations carried by each cell line are indicated below the graphs. A plus sign indicates the absence of a mutation. Results were normalized to GAPDH expression, and asterisks represent significant differences (*p < 0.5). Data are expressed as mean ± SEM; n = 3.

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References

    1. Berger W., Kloeckener-Gruissem B., Neidhardt J. The molecular basis of human retinal and vitreoretinal diseases. Prog. Retin. Eye Res. 2010;29:335–375. - PubMed
    1. McGee T.L., Seyedahmadi B.J., Sweeney M.O., Dryja T.P., Berson E.L. Novel mutations in the long isoform of the USH2A gene in patients with Usher syndrome type II or non-syndromic retinitis pigmentosa. J. Med. Genet. 2010;47:499–506. - PMC - PubMed
    1. Kimberling W.J., Hildebrand M.S., Shearer A.E., Jensen M.L., Halder J.A., Trzupek K., Cohn E.S., Weleber R.G., Stone E.M., Smith R.J. Frequency of Usher syndrome in two pediatric populations: Implications for genetic screening of deaf and hard of hearing children. Genet. Med. 2010;12:512–516. - PMC - PubMed
    1. Millán J.M., Aller E., Jaijo T., Blanco-Kelly F., Gimenez-Pardo A., Ayuso C. An update on the genetics of Usher syndrome. J. Ophthalmol. 2011;2011:417217. - PMC - PubMed
    1. Yan D., Liu X.Z. Genetics and pathological mechanisms of Usher syndrome. J. Hum. Genet. 2010;55:327–335. - PMC - PubMed

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