Generation of CRB1 RP Patient-Derived iPSCs and a CRISPR/Cas9-Mediated Homology-Directed Repair Strategy for the CRB1 c.2480G>T Mutation

Bruna Lopes da Costa; Yao Li; Sarah R Levi; Stephen H Tsang; Peter M J Quinn

doi:10.1007/978-3-031-27681-1_83

Generation of CRB1 RP Patient-Derived iPSCs and a CRISPR/Cas9-Mediated Homology-Directed Repair Strategy for the CRB1 c.2480G>T Mutation

Adv Exp Med Biol. 2023:1415:571-576. doi: 10.1007/978-3-031-27681-1_83.

Authors

Bruna Lopes da Costa^{1

2

3}, Yao Li^{2

3}, Sarah R Levi^{2

3}, Stephen H Tsang^{1

2

3

4

5

6}, Peter M J Quinn^{7

8}

Affiliations

¹ Department of Biomedical Engineering, Columbia University, New York, NY, USA.
² Edward S. Harkness Eye Institute, Department of Ophthalmology, Columbia University Irving Medical Center/New York-Presbyterian Hospital, New York, NY, USA.
³ Jonas Children's Vision Care, and Bernard & Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, Columbia University, New York, NY, USA.
⁴ Columbia Stem Cell Initiative, Columbia University, New York, NY, USA.
⁵ Department of Pathology & Cell Biology, Columbia University, New York, NY, USA.
⁶ Institute of Human Nutrition, Columbia University, New York, NY, USA.
⁷ Edward S. Harkness Eye Institute, Department of Ophthalmology, Columbia University Irving Medical Center/New York-Presbyterian Hospital, New York, NY, USA. pq2138@cumc.columbia.edu.
⁸ Jonas Children's Vision Care, and Bernard & Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, Columbia University, New York, NY, USA. pq2138@cumc.columbia.edu.

PMID: 37440088
DOI: 10.1007/978-3-031-27681-1_83

Abstract

Mutations in the Crumbs-homologue-1 (CRB1) gene lead to a spectrum of severe inherited retinal diseases, including retinitis pigmentosa (RP). The establishment of a genotype-phenotype correlation in CRB1 patients has been difficult due to the substantial variability and phenotypic overlap between CRB1-associated diseases. This phenotypic modulation may be due to several factors, including genetic modifiers, deep intronic mutations, isoform diversity, and copy number variations. Induced pluripotent stem cell (iPSC)-derived patient retinal organoids are novel tools that can provide sensitive, quantitative, and scalable phenotypic assays. CRB1 RP patient iPSC-derived retinal organoids have shown reproducible phenotypes compared to healthy retinal organoids. However, having genetically defined iPSC isogenic controls that take into account potential phenotypic modulation is crucial. In this study, we generated iPSC from an early-onset CRB1 patient and developed a correction strategy for the c.2480G>T, p.(Gly827Val) CRB1 mutation using CRISPR/Cas9-mediated homology-directed repair.

Keywords: Crumbs-homologue-1 (CRB1); Gene editing; Homology-directed repair (HDR); Retinitis pigmentosa (RP); iPSC-derived retinal organoids.

MeSH terms

CRISPR-Cas Systems / genetics
DNA Copy Number Variations
Eye Proteins / genetics
Humans
Induced Pluripotent Stem Cells*
Membrane Proteins / genetics
Mutation
Nerve Tissue Proteins / genetics
Retinitis Pigmentosa* / genetics
Retinitis Pigmentosa* / therapy

Substances

Eye Proteins
CRB1 protein, human
Membrane Proteins
Nerve Tissue Proteins