CRISPR-AsCas12a Efficiently Corrects a GPR143 Intronic Mutation in Induced Pluripotent Stem Cells from an Ocular Albinism Patient

Abstract

Mutations in the GPR143 gene cause X-linked ocular albinism type 1 (OA1), a disease that severely impairs vision. We recently generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of an OA1 patient carrying a point mutation in intron 7 of GPR143. This mutation activates a new splice site causing the incorporation of a pseudoexon. In this study, we present a high-performance CRISPR-Cas ribonucleoprotein strategy to permanently correct the GPR143 mutation in these patient-derived iPSCs. Interestingly, the two single-guide RNAs available for SpCas9 did not allow the cleavage of the target region. In contrast, the cleavage achieved with the CRISPR-AsCas12a system promoted homology-directed repair at a high rate. The CRISPR-AsCas12a-mediated correction did not alter iPSC pluripotency or genetic stability, nor did it result in off-target events. Moreover, we highlight that the disruption of the pathological splice site caused by CRISPR-AsCas12a-mediated insertions/deletions also rescued the normal splicing of GPR143 and its expression level.

FIG. 1.

CRISPR-SpCas9 targets and sgRNA screening in OA1 patient-derived iPSCs. (A) Sequence of the double-stranded GPR143 intron 7 surrounding the patient's G > A mutation (highlighted in red). The sequence of the two tested sgRNAs is also shown, with the 3′ PAM sequence underlined. (B) In vitro digestion assay for the positive control (C+) sgRNA targeting the CDC42BPB gene. The ND PCR product size is 478 bp, whereas the estimated sizes of the CRISPR-SpCas9-digested bands are 407 and 71 bp, with only the higher band visible in the agarose gel. (C) In vitro digestion assay for the sgRNAs targeting the GPR143 intronic mutation. The ND PCR product size is 576 bp, whereas the estimated sizes of the CRISPR-SpCas9-digested bands are ∼315 and ∼261 bp. (D) Surveyor assay of OA1 patient-derived iPSCs treated with the C+ sgRNA targeting the CDC42BPB gene, and (E) with the sgRNAs targeting the GPR143 intronic mutation. NT cells were used as control. S- and S+, samples incubated in the absence or presence of the Surveyor nuclease, respectively. (F) Percentage of indels in the CRISPR-SpCas9-treated patient-derived iPSCs estimated by the TIDE and the ICE analyses. iPSCs, induced pluripotent stem cells; MW, molecular-weight ladder; ND, non-digested; NT, non-treated; OA1, ocular albinism type 1; PAM, protospacer adjacent motif; PCR, polymerase chain reaction; sgRNA, single-guide gRNA; TIDE, Tracking of Indels by DEcomposition.

FIG. 2.

CRISPR-AsCas12a targets and crRNA screening in OA1 patient-derived iPSCs. (A) Sequence of the double-stranded GPR143 intron 7 surrounding the patient's G > A mutation (highlighted in red). The sequence of the four tested crRNAs is also shown, with the 5′ PAM sequence underlined. (B) In vitro digestion assay for the positive control (C+) crRNA targeting the hHPRT1 gene. The ND PCR product size is 1,083 bp, whereas the estimated sizes of the CRISPR-AsCas12a-digested bands are 570 and 513 bp. (C) In vitro digestion assay for the four crRNAs targeting the GPR143 intronic mutation. The ND PCR product size is 576 bp, whereas the estimated sizes of the CRISPR-AsCas12a-digested bands are 312 and 264 bp for crRNA-1, 304 and 276 bp for crRNA-2, 340 and 236 bp for crRNA-3, and 331 and 245 bp for crRNA-4. (D) Surveyor assay of OA1 patient-derived iPSCs treated with the C+ crRNA targeting the hHPRT1 gene and (E) with the four crRNAs targeting the GPR143 intronic mutation. NT cells were used as control. S- and S+, samples incubated in the absence or presence of the Surveyor nuclease, respectively. (F) Percentage of indels in the CRISPR-AsCas12a-treated patient-derived iPSCs estimated by the TIDE analysis. crRNA, CRISPR gRNA.

FIG. 3.

CRISPR-AsCas12a-mediated HDR of the GPR143 intron 7 mutation in OA1 patient-derived iPSCs. (A) Sequence of the double-stranded GPR143 intron 7 surrounding the patient's G > A mutation, which is highlighted in red. The sequence of the two selected crRNAs is also shown, with the 5′ PAM sequence underlined. The 88 nucleotide-long ssODN donor template used for the HDR experiment is shown in the lower part of the panel, with the correct nucleotide highlighted in green. (B) Percentage of indels (dark gray bars) and HDR events (light gray bars) detected in the CRISPR-AsCas12a-treated patient-derived iPSCs by the TIDE and Tracking of Insertion, Deletions, and Recombination (TIDER) events analyses, respectively. (C) Pie chart representing the proportion of the CRISPR-AsCas12a + ssODN-induced modification categories in the 14 clones surviving after single cell sorting. (D) Detailed sequence analysis of the 14 surviving clones after CRISPR-AsCas12a + ssODN-treatment. The patient's mutation is highlighted in red and the correct nucleotide in green. The sequence of control cells (WT) was used for comparison, and differences are highlighted with a # symbol. HDR, homologous-directed repair; ssODN, single-stranded oligodeoxynucleotide; WT, wild-type.

FIG. 4.

Effect of the correction of the GPR143 intron 7 mutation in OA1 patient-derived iPSCs on splicing and expression. (A) Evaluation of the GPR143 transcript size via reverse transcription (RT)-PCR analysis. The normal mRNA amplicon from exons 6 to 8 is 330 bp long, as shown in healthy control cells (WT) and edited clones. The intron 7 mutation causes the incorporation of a 165-bp-long pseudoexon, resulting in the predominant 495-bp-long band (upper band) seen in the NT patient-derived iPSCs; a barely visible normal-sized band (lower band) also can be detected. GAPDH transcript amplification was used as loading control. C-, negative control. (B) GPR143 expression levels, evaluated by quantitative PCR analysis, in healthy control iPSCs (WT, light gray bar), NT (black bar), and CRISPR-AsCas12a + ssODN-treated (fully corrected clones in light gray bars and clones presenting indels in dark gray bars) patient-derived iPSCs. Results were normalized to GAPDH expression, and WT cells were used as reference by setting at 1 their relative expression. ***Highlight statistically significant difference (p ≤ 0.0001) of NT sample against all the other samples. Data are expressed as mean ± SEM, n = 3.

FIG. 5.

Characteristic stem cell phenotype and genetic stability of the CRISPR-AsCas12a-corrected OA1 iPSC clones A8 and B10. Phase-contrast image of growing colonies for clone A8 (A) and B10 (K). Scale bars = 200 μm. KaryoStat microarray analysis for the genetic integrity evaluation of clone A8 (B) and B10 (L). The whole-genome view displays all somatic and sex chromosomes (X-axis), with the pink, green, and yellow colors indicating the raw signal for each individual chromosome probe. The blue line represents the normalized probe signal that is used to identify copy number and aberrations, if any (Y-axis). A value of 2 represents a normal copy number condition (CN = 2), whereas 3 indicates chromosomal gain (CN = 3) and 1 a chromosomal loss (CN = 1). A value of 1 for both the X and the Y chromosomes indicates that the sample originated from a male individual. Green immunostaining of the typical pluripotency markers NANOG (C, M), OCT4 (D, N), SOX2 (E, O), and TRA1–81 (F, P) in A8 and B10 clones, respectively. Nuclei are DAPI-labeled in blue. Scale bars = 50 μm. Green immunostaining following in vitro differentiation of the specific germ layer markers OTX2 for ectoderm (G, Q), BRACHYURY for mesoderm (H, R), and SOX17 for endoderm (I, S) in A8 and B10 clones, respectively. Nuclei are DAPI-labeled in blue, and actin is phalloidin-labeled in red. Scale bars = 50 μm. Cell ID correlation plot for clones A8 (J) and B10 (T). 150k single-nucleotide polymorphisms spread across the genome were analyzed and compared between samples. NT patient-derived iPSCs were used as control reference sample. The correlation between a sample and itself gives a value of 100%; correlations >95% between samples indicate that they have an identical genetic background. CN, copy number; DAPI, 4′,6-diamidino-2-phenylindole.