Using CRISPR-Cas9 to Generate Gene-Corrected Autologous iPSCs for the Treatment of Inherited Retinal Degeneration

Mol Ther. 2017 Sep 6;25(9):1999-2013. doi: 10.1016/j.ymthe.2017.05.015. Epub 2017 Jun 12.


Patient-derived induced pluripotent stem cells (iPSCs) hold great promise for autologous cell replacement. However, for many inherited diseases, treatment will likely require genetic repair pre-transplantation. Genome editing technologies are useful for this application. The purpose of this study was to develop CRISPR-Cas9-mediated genome editing strategies to target and correct the three most common types of disease-causing variants in patient-derived iPSCs: (1) exonic, (2) deep intronic, and (3) dominant gain of function. We developed a homology-directed repair strategy targeting a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) and demonstrated restoration of the retinal transcript and protein in patient cells. We generated a CRISPR-Cas9-mediated non-homologous end joining (NHEJ) approach to excise a major contributor to Leber congenital amaurosis, the IVS26 cryptic-splice mutation in CEP290, and demonstrated correction of the transcript and protein in patient iPSCs. Lastly, we designed allele-specific CRISPR guides that selectively target the mutant Pro23His rhodopsin (RHO) allele, which, following delivery to both patient iPSCs in vitro and pig retina in vivo, created a frameshift and premature stop that would prevent transcription of the disease-causing variant. The strategies developed in this study will prove useful for correcting a wide range of genetic variants in genes that cause inherited retinal degeneration.

Keywords: CRISPR; iPSCs; retinal degeneration.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Animals
  • CRISPR-Cas Systems*
  • Cell Line
  • Gene Editing*
  • Gene Order
  • Gene Targeting*
  • Genetic Loci
  • Genetic Therapy
  • Genetic Vectors / genetics
  • Homologous Recombination
  • Humans
  • Induced Pluripotent Stem Cells / cytology
  • Induced Pluripotent Stem Cells / metabolism*
  • Introns
  • Mutation
  • Protein-Serine-Threonine Kinases / genetics
  • RNA, Guide
  • Retinal Degeneration / genetics*
  • Retinal Degeneration / therapy
  • Stem Cell Transplantation* / methods
  • Transplantation, Autologous


  • RNA, Guide
  • Protein-Serine-Threonine Kinases
  • MAK protein, human