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

• Published in: Scientific reports Vol. 13; no. 1; p. 3142
• Main Authors: Lu, Fangfang, Leach, Lyndsay L., Gross, Jeffrey M.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 23.02.2023; Nature Publishing Group; Nature Portfolio
• 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
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-023-29046-5

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.