Large-scale CRISPR screen reveals context-specific genetic regulation of retinal ganglion cell regeneration.

Many genes are known to regulate retinal regeneration following widespread tissue damage. Conversely, genes controlling regeneration following limited cell loss, per degenerative diseases, are undefined. As stem/progenitor cell responses scale to injury levels, understanding how the extent and specificity of cell loss impact regenerative processes is important. Here, transgenic zebrafish enabling selective retinal ganglion cell (RGC) ablation were used to identify genes that regulate RGC regeneration. A single cell multiomics-informed screen of 101 genes identified seven knockouts that inhibited and eleven that promoted RGC regeneration. Surprisingly, 35 of 36 genes known/implicated as being required for regeneration following widespread retinal damage were not required for RGC regeneration, and seven even enhanced regeneration kinetics, including proneural factors neurog1, olig2, and ascl1a. Mechanistic analyses revealed ascl1a disruption increased the propensity of progenitor cells to produce RGCs; i.e., increased "fate bias". These data demonstrate plasticity in how Müller glia can convert to a stem-like state and context-specificity in how genes function during regeneration. Increased understanding of how the regeneration of disease-relevant cell types is specifically controlled will support the development of disease-tailored regenerative therapeutics.

Molecular regulation of retinal regeneration is context specific.

Many genes are known to regulate retinal regeneration following widespread tissue damage. Conversely, genes controlling regeneration following limited retinal cell loss, akin to disease conditions, are undefined. Combining a novel retinal ganglion cell (RGC) ablation-based glaucoma model, single cell omics, and rapid CRISPR/Cas9-based knockout methods to screen 100 genes, we identified 18 effectors of RGC regeneration kinetics. Surprisingly, 32 of 33 previously known/implicated regulators of retinal tissue regeneration were not required for RGC replacement; 7 knockouts accelerated regeneration, including sox2, olig2, and ascl1a . Mechanistic analyses revealed loss of ascl1a increased "fate bias", the propensity of progenitors to produce RGCs. These data demonstrate plasticity and context-specificity in how genes function to control regeneration, insights that could help to advance disease-tailored therapeutics for replacing lost retinal cells. One sentence summary: We discovered eighteen genes that regulate the regeneration of retinal ganglion cells in zebrafish.