Table 2.
Applications of CRISPR/Cas9 technologies combined with iPSCs in the study and treatment of retinal degenerative diseases.
| Publication | iPSC Source | Retinal Disease | Gene; Mutation; Patient Information | CRISPR-Mediated Genome Editing: Mechanism and Delivery |
|---|---|---|---|---|
| Bassuk et al., 2016 [98] | Dermal skin fibroblasts | XLRP | RPGR c.3070G>T | HDR correction of mutation. Transfection with plasmid for SpCas9 and gRNA expression and ssODN template. |
| Burnight et al., 2017 [107] | Dermal skin fibroblasts | RP, LCA10 | 353-bp Alu insertion in MAK (Patient 1) CEP290 c.2991 + 1655 A>G, IVS26 (Patient 2–4) RHO c.163 C>A (Patient 5) |
HDR correction of mutation 353-bp Alu insertion in MAK, by nucleofection with plasmids for SpCas9 and gRNA expression and HDR template. NHEJ-mediated repair and HDR correction of IVS26 mutation, by transfection of plasmids for SpCas9 or SaCas9 and single or dual gRNA expression. Knockout and HDR correction of RHO c.163 C>A. |
| Buskin et al., 2018 [102] | Dermal skin fibroblasts | RP11 |
PRPF31 c.1115_1125 del11 (Patient 1–3) PRPF31 c.522_527+10del (Patient 4) |
HDR correction of c.1115_1125 del11 mutation in a patient-specific iPSC line. Transfection of plasmid for SpCas9 and gRNA expression and ssODN template. |
| Deng et al., 2018 [99] | Renal epithelial cells | XLRP |
RPGR c.1685_1686delAT (Patient 1) RPGR c.2234_2235delGA (Patient 2) RPGR c.2403_2404delAG (Patient 3) |
HDR correction of c.1685_1686delAT mutation. Nucleofection with plasmids for SpCas9 and gRNA expression and HDR template. |
| Foltz et al., 2018 [100] | Dermal skin fibroblasts | RP13 | PRPF8 c.6901C>T | HDR correction of mutation. Electroporation with plasmids for gRNA expression, Cas9-Gem mRNA and ssODN template. |
| Gagliardi et al., 2018 [120] | Retinal Müller glial cells | / | AAVS1 | CRISPR-mediated HDR to introduce a reporter gene under the control of murine Crx promoter, in AAVS1 site. Transfection with plasmids for SpCas9 and gRNA expression and HDR template. |
| Artero-Castro et al., 2019 [104] | Dermal skin fibroblasts | arRP | MERTK c.992_993delCA | HDR correction of mutation. Transfection with eSpCas9_1.1 RNP and ssODN template. Clones carrying homozygous and heterozygous correction were obtained. |
| Bohrer et al., 2019 [108] | Dermal skin fibroblasts | ESCS |
NR2E3 c.119-2A>C (Patient 1) NR2E3 c.219G>C and c.932G>A (Patient 2) |
HDR correction of c.119-2A>C (Patient 1) and c.219G>C (Patient 2) mutations. Transfection with plasmids for SpCas9 and gRNA expression and HDR template. |
| Brydon et al., 2019 [115] | Dermal skin fibroblasts | RP11 |
PRPF31 exon 7 (Healthy donor) PRPF31 c.1115_1125del11(Patient) |
Knockout of PRPF31 by targeting exon 7 in wt iPSC to create a haploinsufficient PRPF31+/- line, by nucleofection with plasmids for SpCas9 and gRNA expression. RP11-iPSC line carrying c.1115_1125 del11 mutation and corrected by CRISPR-mediated HDR (Buskin et al., 2018). |
| Huang et al., 2019 [106] | PBMC | XLRS |
RS1 c.625C>T (Patient 1) RS1 c.488G>A (Patient 2) |
Correction of c.625C>T mutation in patient iPSC by CRISPR-mediated HDR and also an adenine base editing approach. Introduction of c.625C>T mutation in wt iPSC. Nucleofection of plasmids for Cas9, gRNA and HDR templates and ABE7.10 base editor. |
| Kanzaki et al., 2020 [29] | Dental pulp cells | LCA16 | KCNJ13 | Knockout of KCNJ13 by targeting exon 2 and 3 in wt iPSC. Electroporation with SpCas9 protein and gRNA expression. |
| Lam et al., 2020 [117] | Dermal skin fibroblasts | / |
VSX2
BRN3b RCVRN |
CRISPR-mediated HDR to introduce reporter genes in targeted loci. Transfection with plasmids for SpCas9 and gRNA expression and HDR templates. |
| Lane et al., 2020 [116] | Dermal skin fibroblasts | XLRP |
RP2 exon 2 RP2 c.358C>T |
Knockout of RP2 by targeting exon 2 in wt iPSC. Electroporation with plasmid for SpCas9 and gRNA expression. |
| Sanjurjo-Soriano et al., 2020 [109] | Dermal skin fibroblasts | USH2A |
USH2A c.2299delG (Patient 1) USH2A c.2276G>T and c.2299delG (Patient 2) |
HDR correction of USH2A mutations. Nucleofection with plasmid for eSpCas9(1.1) and ssODN template. |
| Yang et al., 2020 [114] | PBMC | XLRS | RS1 | Introduction of RS1 c.625C>T in wt iPSC. Nanodiamond carriers of linear DNA for CRISPR components and HDR template. |
| Artero-Castro et al., 2021 [105] | Dermal skin fibroblasts | arRP | MERTK c.992_993delCA | iPSC lines carrying c.992_993delCA mutation and homozygously or heterozygously corrected by CRISPR-mediated HDR (Artero-Castro et al., 2019). |
| Chirco et al., 2021 [113] | PBMC | LCA7 |
CRX c.464_465insGGCA CRX c.262A>C |
Knockout of CRX c.262A>C mutated allele. Transfection with plasmids for SpCas9 and gRNA expression. |
| Diakatou et al., 2021 [112] | Dermal skin fibroblasts | adRP | NR2E3 c.166G>A | Knockout of NR2E3 mutated allele. Transfection with plasmid for SpCas9 and mutation-specific gRNA expression. |
| Liu et al., 2021 [110] | PBMC | USH2A | USH2A c.2299delG and c.1256G>T | HDR correction of USH2A c.2299delG mutation. Transfection with SpCas9 RNP and ssODN template. |
| Matsuyama et al., 2021 [119] | Murine fibroblasts | / |
Bhlhb4
Islet1 |
Knockout of Bhlhb4 and Islet1 in Tg(Nrl-GFP); Ribeye-reporter miPS cells and Thy1-GCaMP6f; Ribeye-reporter mES cells. Nucleofection with plasmids for SpCas9 and gRNA expression. |
| Wahlin et al, 2021 [118] | Fibroblasts | / |
SIX6
POU4F2 |
CRISPR-mediated HDR to introduce reporter genes in targeted loci. Transfection with plasmids for SpCas9 and gRNA expression and HDR templates. |
| Cheng et al., 2022 [97] | Dermal skin fibroblasts | ODDD | CX43 | Knockout of CX43 in wt iPSC. Nucleofection with plasmid for SpCas9 protein and gRNA expression. |
| Guan et al., 2022 [121] | Bone marrow CD34+ cells | / | RCVRN | CRISPR-mediated HDR to introduce a reporter gene in the targeted locus. Transfection with plasmids for SpCas9 and gRNA expression and HDR template. |
| Georgiou et al., 2022 [103] | Dermal skin fibroblasts | RP11 |
PRPF31 c.1115_1125 del11 (Patient 1–3) c.522_527+10del (Patient 4) |
RP11-iPSC line carrying c.1115_1125 del11 mutation and corrected by CRISPR-mediated HDR (Buskin et al., 2018). |
| Leung et al., 2022 [111] | Renal epithelial cells | LCA4 |
AIPL1 c.834G>A (Patients 1) AIPL1 c.834G>A and c.466-1G>C (Patients 2–3) AIPL1 c.834G>A and c.665G>A (Patients 4) |
HDR correction of mutation to create isogenic control lines. Nucleofection with eSpCas9_1.1 RNP and ssODN template. |