Phenotypic screening using synthetic CRISPR gRNAs reveals pro-regenerative genes in spinal cord injury
Phenotypic screening using synthetic CRISPR gRNAs reveals pro-regenerative genes in spinal cord injury
复制标题
DOI：
10.1101/2020.04.03.023119
复制
发表时间：
2020-04
期刊：
影响因子：
--
通讯作者：
Marcus Keatinge;Themistoklis M Tsarouchas;Tahimina Munir;J. Larraz;Davide Gianni;Hui-hsin Tsai;C. G. Becker;D. Lyons;T. Becker
中科院分区：
文献类型：
--
作者：
Marcus Keatinge;Themistoklis M Tsarouchas;Tahimina Munir;J. Larraz;Davide Gianni;Hui-hsin Tsai;C. G. Becker;D. Lyons;T. Becker
急性CRISPR/Cas9靶向为斑马鱼中可扩展的表型遗传筛选提供了机会。然而，CRISPR gRNA（CrRNA）活性的不可预测的效率是一个限制因素。在这里，我们描述了如何解决这一问题，通过预筛选CrRNA在体内的高活性，使用一个简单的标准化分析的基础上限制性片段长度多态性分析（RFLP）。我们在斑马鱼胚胎中用合成的RNA Oligo guide RNA（sCrRNA）靶向350个基因组位点，发现几乎一半在我们的RFLP测定中表现出> 90%的效率。有能力预选高活性的sCrRNA（haCRs），我们进行了集中的表型筛选的30个巨噬细胞相关基因在脊髓再生，发现10个基因的破坏损害轴突再生。随后分析的5个稳定突变体中有4个（tgfb 1a、tgfb 3、tnfa、sparc）保留了急性haCR表型，验证了这种方法的效率。从机制上讲，缺乏tgfb 1a会导致损伤后免疫反应延长，从而抑制再生。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，并可应用于研究任何感兴趣的生物功能。亮点-合成的CRISPR gRNA具有高度活性-体内预筛选可快速评估CRISPR gRNA活性-表型CRISPR筛选揭示了脊髓再生的关键基因-tgfb 1a通过控制炎症促进脊髓再生
Acute CRISPR/Cas9 targeting offers the opportunity for scalable phenotypic genetic screening in zebrafish. However, the unpredictable efficiency of CRISPR gRNA (CrRNA) activity is a limiting factor. Here we describe how to resolve this by prescreening CrRNAs for high activity in vivo, using a simple standardised assay based on restriction fragment length polymorphism analysis (RFLP). We targeted 350 genomic sites with synthetic RNA Oligo guide RNAs (sCrRNAs) in zebrafish embryos and found that almost half exhibited > 90% efficiency in our RFLP assay. Having the ability to preselect highly active sCrRNAs (haCRs), we carried out a focussed phenotypic screen of 30 macrophage-related genes in spinal cord regeneration and found 10 genes whose disruption impaired axonal regeneration. Four (tgfb1a, tgfb3, tnfa, sparc) out of 5 stable mutants subsequently analysed retained the acute haCR phenotype, validating the efficiency of this approach. Mechanistically, lack of tgfb1a leads to a prolonged immune response after injury, which inhibits regeneration. Our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, and can be applied to study any biological function of interest. HIGHLIGHTS - Synthetic CRISPR gRNAs are highly active - in vivo pre-screening allows rapid assessment of CRISPR gRNA activity - Phenotypic CRISPR screen reveals crucial genes for spinal cord regeneration - tgfb1a promotes spinal regeneration by controlling inflammation