Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA.
Department of Ophthalmology, The Second Xiangya Hospital, Central South University, Changsha, 410011, Hunan, China.
Sci Rep. 2023 Feb 23;13(1):3142. 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.
导致视网膜色素上皮 (RPE) 细胞死亡的眼部疾病会导致视力丧失和失明。目前，还没有获得 FDA 批准的策略来修复受损的 RPE 细胞。刺激受损组织中的内在再生反应已成为组织修复的一种可能机制。斑马鱼具有出色的再生能力，包括 RPE 在内；然而，我们对其潜在机制的理解仍然有限。在这里，我们对 27 个候选 RPE 再生基因进行了 F0 体内 CRISPR-Cas9 介导的筛选。该筛选涉及注射含有每个靶基因三个高诱变向导 RNA 的核糖核蛋白复合物，然后进行基于 PCR 的基因分型，以鉴定大的基因内缺失，并使用 MATLAB 进行基于自动化的 RPE 再生定量。通过该 F0 筛选流程，鉴定出了 8 个 RPE 再生的阳性和 7 个阴性调节因子。对一个候选基因 cldn7b 的进一步表征揭示了其在 RPE 损伤后调节巨噬细胞/小胶质细胞浸润以及在晚期 RPE 再生过程中清除 RPE/色素碎片方面的新作用。综上所述，这些数据支持靶向 F0 筛选用于验证促再生因子的有效性，并揭示了可能调节斑马鱼 RPE 再生反应的新因子。
