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A CRISPR-Cas9-mediated F0 screen to identify pro-regenerative genes in the zebrafish retinal pigment epithelium
Fangfang Lu1,2, Lyndsay L Leach1,3, Jeffrey M Gross4,5
1Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA.
Abstract:
Ocular diseases resulting in death of the retinal pigment epithelium (RPE) lead to vision loss and blindness. There are currently no FDA-approved strategies to restore damaged RPE cells. Stimulating intrinsic regenerative responses within damaged tissues has gained traction as a possible mechanism for tissue repair. Zebrafish possess remarkable regenerative abilities, including within the RPE; however, our understanding of the underlying mechanisms remains limited. Here, we conducted an F0 in vivo CRISPR-Cas9-mediated screen of 27 candidate RPE regeneration genes. The screen involved injection of a ribonucleoprotein complex containing three highly mutagenic guide RNAs per target gene followed by PCR-based genotyping to identify large intragenic deletions and MATLAB-based automated quantification of RPE regeneration. Through this F0 screening pipeline, eight positive and seven negative regulators of RPE regeneration were identified. Further characterization of one candidate, cldn7b, revealed novel roles in regulating macrophage/microglia infiltration after RPE injury and in clearing RPE/pigment debris during late-phase RPE regeneration. Taken together, these data support the utility of targeted F0 screens for validating pro-regenerative factors and reveal novel factors that could regulate regenerative responses within the zebrafish RPE.
Insights
Researchers identified key genes that regulate retinal pigment epithelium (RPE) regeneration in zebrafish using CRISPR-Cas9 screening. This study reveals new factors that could potentially restore vision loss caused by RPE damage.
Area of Science:
- Ophthalmology
- Regenerative Medicine
- Genetics
Background:
- Ocular diseases causing retinal pigment epithelium (RPE) death lead to irreversible vision loss.
- Current treatments lack FDA-approved strategies for RPE cell restoration.
- Zebrafish exhibit significant regenerative capabilities, offering a model to study RPE repair mechanisms.
Purpose of the Study:
- To identify genes involved in RPE regeneration using a large-scale genetic screen.
- To validate potential pro-regenerative factors for therapeutic applications.
- To uncover novel mechanisms regulating RPE repair in zebrafish.
Main Methods:
- Conducted an F0 in vivo CRISPR-Cas9-mediated screen of 27 candidate RPE regeneration genes.
- Utilized ribonucleoprotein complexes with mutagenic guide RNAs and PCR-based genotyping.
- Employed MATLAB-based automated quantification to assess RPE regeneration efficiency.
Main Results:
- Identified eight positive and seven negative regulators of RPE regeneration.
- Characterized cldn7b, revealing its role in macrophage/microglia infiltration and debris clearance during RPE regeneration.
- Demonstrated the effectiveness of targeted F0 screens for discovering regenerative factors.
Conclusions:
- Targeted F0 screens are a valuable tool for identifying and validating pro-regenerative factors.
- Novel factors, such as cldn7b, play critical roles in the complex process of RPE regeneration.
- Findings provide insights into potential therapeutic strategies for vision loss due to RPE damage.
