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

Abstract

ABSTRACTOcular 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.Summary statementWe present a rapid CRISPR-Cas9-mediated F0 screen, which revealed novel regulators of retinal pigment epithelium (RPE) regeneration in zebrafish. This screen and identified factors will greatly facilitate discovery of underlying RPE regenerative mechanisms.