A CRISPR-Cas9-mediated F0 screen to identify pro-regenerative genes in the zebrafish retinal pigment epithelium.
A CRISPR-Cas9-mediated F0 screen to identify pro-regenerative genes in the zebrafish retinal pigment epithelium.
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DOI：
10.1038/s41598-023-29046-5
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发表时间：
2023-02-23
影响因子：
4.6
通讯作者：
中科院分区：
文献类型：
--
作者：
眼病导致视网膜色素上皮（RPE）死亡，导致视力丧失和失明。目前还没有fda批准的策略来恢复受损的RPE细胞。刺激受损组织内的内在再生反应已成为组织修复的可能机制。斑马鱼具有非凡的再生能力，包括在RPE内；然而，我们对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。筛选包括为每个靶基因注射含有三种高度诱变引导rna的核糖核蛋白复合物，然后进行基于pcr的基因分型，以鉴定基因内的大缺失，并基于matlab的RPE再生自动量化。通过F0筛选管道，鉴定出8个RPE再生阳性调节因子和7个RPE再生阴性调节因子。进一步表征一种候选cldn7b，揭示了在RPE损伤后调节巨噬细胞/小胶质细胞浸润以及在后期RPE再生过程中清除RPE/色素碎片的新作用。综上所述，这些数据支持了靶向F0筛选用于验证促再生因子的效用，并揭示了可以调节斑马鱼RPE内再生反应的新因子。
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.
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影响因子：
7.8
作者：
Chen M;Rajapakse D;Fraczek M;Luo C;Forrester JV;Xu H
通讯作者：
Xu H
影响因子：
4.5
作者：
Keatinge M;Tsarouchas TM;Munir T;Porter NJ;Larraz J;Gianni D;Tsai HH;Becker CG;Lyons DA;Becker T
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Becker T
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Hanovice, Nicholas J.;Leach, Lyndsay L.;Gross, Jeffrey M.
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Gross, Jeffrey M.
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作者：
Unal Eroglu A;Mulligan TS;Zhang L;White DT;Sengupta S;Nie C;Lu NY;Qian J;Xu L;Pei W;Burgess SM;Saxena MT;Mumm JS
通讯作者：
Mumm JS
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F