CRISPR Interference–Potential Application in Retinal Disease
Abstract
:1. Introduction
2. Opportunities for Gene Regulation in Eukaryotes
3. Mechanisms of CRISPRi
4. Alternatives to CRISPRi
4.1. CRISPRi vs. CRISPR/Cas9
4.2. CRISPRi vs. RNAi
5. Clinical Treatment Strategies
5.1. In Vivo Knock-Down
5.1.1. Targeting the Pathogenic Mutation
5.1.2. Cellular Reprogramming
5.1.3. Treating Disease Pathways
5.2. Ex Vivo Knock-Down
6. Challenges to Clinical Application
6.1. Target Selection and Efficacy
6.2. Delivery to the Retina
6.3. Immune Response
7. Conclusions
Author Contributions
Funding
Conflicts of Interest
Abbreviations
AAV | Adeno-associated virus |
CRISPRi | CRISPR interference |
gRNA | Guide RNA |
iPSCs | Induced pluripotent stem cells |
KRAB | Krüppel associated box |
RNAi | RNA interference |
RNP | Ribonucleoprotein |
SNP | Single nucleotide polymorphism |
TSS | Transcriptional start site |
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Gene Regulation | Method |
---|---|
Gene transcription | CRISPRi |
DNA methylation | dCas9-methyltransferase |
mRNA lifespan/gene translation | RNAi |
Histone state | CRISPRi |
Feature | CRISPRi | CRISPR/Cas9 | RNAi |
---|---|---|---|
Target | DNA | DNA | mRNA |
Requirements | gRNA complementary to target. dCas9 protein | gRNA complementary to target. Cas9 protein | Short interfering RNA complementary to target |
Efficiency | + | ++ | + |
Specificity | ++ | + | + |
Reference | Treatment Method | Condition | gRNA Target | Experimental Methodology | Results |
---|---|---|---|---|---|
Moreno et al. 2018 [48] | In vivo knock-down Cellular reprogramming | Autosomal recessive retinitis pigmentosa | Nrl | Dual subretinal injection of AAV.gRNA.dSpCas9-KRAB.N-terminus and AAV.dSpCas9-KRAB C-terminus into Rd10 mouse. | Rods developed a more “cone-like” phenotype. Increased photoreceptor layer thickness. Significantly improved visual function. |
Thakore et al. 2018 [54] | In vivo knock-down Treating disease pathways | High LDL cholesterol (familial hypercholesterolemia) | Pcsk9 | Dual injection of AAV.dSaCas9-KRAB and AAV.gRNA into mouse tail vein. | 80% reduction in target protein. Significant reduction in serum LDL cholesterol. |
Chung et al. 2019 [26] | In vivo knock-down Treating disease pathways | Obesity | Fabp4 | Intraperitoneal injection of ATS-9R peptide and dSpCas9.gRNA plasmid oligoplex into HFD-induced obesity and diabetes model mice. | Significant reduction in target mRNA. Improved disease symptoms including decrease in body weight, fat mass, and blood glucose. |
Yoshida et al. 2018 [57] | In vivo knock-down Treating disease pathways (Experimental methodology required ex vivo knock-down) | Lung squamous cell carcinoma | ∆Np63 | Lentiviral delivery of dSpCas9-KRAB.gRNA to EBC2 lung SCC cells. Xenograft then injected into adult mice. | Tumour growth significantly repressed |
Truong et al. 2019 [58] | Ex vivo knock-down | Calvarial bone healing | PPAR-γ (repression) and Sox9 (activation) | Bacilloviral delivery of all-in-one CRISPRai construct into rat bone marrow-derived mesenchymal stem cells. These were implanted into rat calvarial bone defects. | Significant increase in calvarial bone healing. |
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Peddle, C.F.; Fry, L.E.; McClements, M.E.; MacLaren, R.E. CRISPR Interference–Potential Application in Retinal Disease. Int. J. Mol. Sci. 2020, 21, 2329. https://doi.org/10.3390/ijms21072329
Peddle CF, Fry LE, McClements ME, MacLaren RE. CRISPR Interference–Potential Application in Retinal Disease. International Journal of Molecular Sciences. 2020; 21(7):2329. https://doi.org/10.3390/ijms21072329
Chicago/Turabian StylePeddle, Caroline F., Lewis E. Fry, Michelle E. McClements, and Robert E. MacLaren. 2020. "CRISPR Interference–Potential Application in Retinal Disease" International Journal of Molecular Sciences 21, no. 7: 2329. https://doi.org/10.3390/ijms21072329