A CRISPR-Cas9-mediated F0 screen to identify pro-regenerative genes in the zebrafish retinal pigment epithelium

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 f...

Full description

Saved in:
Bibliographic Details
Published in:Scientific reports 2023-02, Vol.13 (1), p.3142-3142, Article 3142
Main Authors: Lu, Fangfang, Leach, Lyndsay L., Gross, Jeffrey M.
Format: Article
Language:English
Subjects:
631/378/1687
692/699/3161/1626
Animals
CRISPR
CRISPR-Cas Systems - genetics
Danio rerio
Epithelium
Genotyping
Humanities and Social Sciences
Macrophages
Microglia
multidisciplinary
Pigments
Retina
Retinal pigment epithelium
Retinal Pigment Epithelium - physiology
Science
Science (multidisciplinary)
Zebrafish - genetics
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-023-29046-5