CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury.
CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury.
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DOI：
10.1371/journal.pgen.1009515
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发表时间：
2021-04
期刊：
影响因子：
4.5
通讯作者：
Becker T
中科院分区：
文献类型：
--
作者：
Keatinge M;Tsarouchas TM;Munir T;Porter NJ;Larraz J;Gianni D;Tsai HH;Becker CG;Lyons DA;Becker T
斑马鱼在脊髓损伤后表现出强大的再生，由控制损伤后炎症的巨噬细胞促进。然而，巨噬细胞如何调节再生的机制基础知之甚少。为了解决这一认识上的差距，我们进行了一个快速的体内表型筛选巨噬细胞相关基因，促进脊髓损伤后再生。我们使用急性注射合成RNA Oligo CRISPR向导RNA（sCrRNA），其在体内预先筛选高活性。对超过350种sCrRNA的预筛选使我们能够快速鉴定出高活性的sCrRNA（多达一半，缩写为haCR），并有效靶向30种潜在的巨噬细胞相关基因。这些基因中的10个的破坏损害了脊髓损伤后的轴突再生。我们选择了5个基因用于进一步分析，并使用haCR产生稳定的突变体。这些突变体中的四个（tgfb 1a、tgfb 3、tnfa、sparc）保留了急性haCR表型，验证了该方法。从机制上讲，tgfb 1a haCR注射和稳定的突变体斑马鱼未能解决损伤后炎症，这表明中性粒细胞的长期存在和il 1b表达水平的增加。抑制IL-1β可挽救tgfb 1a突变体受损的轴突再生因此，我们的快速和可扩展的筛选方法已经确定了脊髓再生的功能调节因子，但可以应用于任何感兴趣的生物功能。在脊髓损伤中被切断的神经连接不会愈合，这可能导致永久性瘫痪。缺乏修复可能部分是由于损伤部位的长期炎症。相比之下，斑马鱼在脊髓损伤后表现出良好的神经连接修复，这与控制炎症有关。由于遗传技术（CRISPR/Cas9）的最新进展，我们现在可以在几天内确定影响斑马鱼再生的基因的功能。在这里，我们设计了一个非常快速的筛选方法，在脊髓损伤后的斑马鱼幼虫的炎症相关基因的功能。我们发现了一些基因，这些基因对于修复神经连接和控制损伤后的炎症是必要的。这提供了重要的线索，以提高我们对炎症在脊髓损伤中的作用的理解。此外，我们的快速和强大的筛选方法可以被其他研究人员采用，以筛选整个动物的基因功能，这在以前是不容易实现的。
Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. We used acute injection of synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) that were pre-screened for high activity in vivo. Pre-screening of over 350 sCrRNAs allowed us to rapidly identify highly active sCrRNAs (up to half, abbreviated as haCRs) and to effectively target 30 potentially macrophage-related genes. Disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. We selected 5 genes for further analysis and generated stable mutants using haCRs. Four of these mutants (tgfb1a, tgfb3, tnfa, sparc) retained the acute haCR phenotype, validating the approach. Mechanistically, tgfb1a haCR-injected and stable mutant zebrafish fail to resolve post-injury inflammation, indicated by prolonged presence of neutrophils and increased levels of il1b expression. Inhibition of Il-1β rescues the impaired axon regeneration in the tgfb1a mutant. Hence, our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, but can be applied to any biological function of interest. Nerve connections that are severed in spinal cord injury do not heal, which can lead to permanent paralysis. Lack of repair may in part be due to prolonged inflammation of the injury site. In contrast, zebrafish show excellent repair of nerve connections after spinal injury and this is associated with controlling inflammation. Due to recent advances in genetic technology (CRISPR/Cas9) we can now determine the function of genes that influence regeneration in the living zebrafish in a matter of days. Here we devise a very rapid screening method for the function of inflammation-related genes in zebrafish larvae after spinal cord injury. We find a number of genes that are necessary for repair of nerve connections and control of the inflammation after injury. This provides important leads to improve our understanding of the role of inflammation in spinal cord injury. Moreover, our fast and robust screening method can be adopted by other researchers to screen for gene functions in a whole animal, which was previously not easily possible.
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影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
6.2
作者：
Charlton-Perkins, Mark;Almeida, Alexandra D.;Harris, William A.
通讯作者：
Harris, William A.
DOI：
10.1073/pnas.1308335110
发表时间：
2013-08-20
影响因子：
11.1
作者：
Jao, Li-En;Wente, Susan R.;Chen, Wenbiao
通讯作者：
Chen, Wenbiao
Keops复合基因的急性多sGrNA敲低重现了稳定的敲除斑马鱼模型的小头畸形表型。
影响因子：
3.7
作者：
Jobst-Schwan T;Schmidt JM;Schneider R;Hoogstraten CA;Ullmann JFP;Schapiro D;Majmundar AJ;Kolb A;Eddy K;Shril S;Braun DA;Poduri A;Hildebrandt F
通讯作者：
Hildebrandt F
影响因子：
4.5
作者：
Anderson JL;Mulligan TS;Shen MC;Wang H;Scahill CM;Tan FJ;Du SJ;Busch-Nentwich EM;Farber SA
通讯作者：
Farber SA