Department of Developmental Biology, Washington University School of Medicine, Saint Louis, MO 63110, USA.
Center of Regenerative Medicine, Washington University School of Medicine, Saint Louis, MO 63110, USA.
G3 (Bethesda). 2021 Aug 7;11(8). doi: 10.1093/g3journal/jkab089.
Adult zebrafish are widely used to interrogate mechanisms of disease development and tissue regeneration. Yet, the prospect of large-scale genetics in adult zebrafish has traditionally faced a host of biological and technical challenges, including inaccessibility of adult tissues to high-throughput phenotyping and the spatial and technical demands of adult husbandry. Here, we describe an experimental pipeline that combines high-efficiency CRISPR/Cas9 mutagenesis with functional phenotypic screening to identify genes required for spinal cord repair in adult zebrafish. Using CRISPR/Cas9 dual-guide ribonucleic proteins, we show selective and combinatorial mutagenesis of 17 genes at 28 target sites with efficiencies exceeding 85% in adult F0 "crispants". We find that capillary electrophoresis is a reliable method to measure indel frequencies. Using a quantifiable behavioral assay, we identify seven single- or duplicate-gene crispants with reduced functional recovery after spinal cord injury. To rule out off-target effects, we generate germline mutations that recapitulate the crispant regeneration phenotypes. This study provides a platform that combines high-efficiency somatic mutagenesis with a functional phenotypic readout to perform medium- to large-scale genetic studies in adult zebrafish.
成年斑马鱼被广泛用于研究疾病发生和组织再生的机制。然而，传统上，对成年斑马鱼进行大规模遗传学研究面临许多生物学和技术挑战，包括难以对成年组织进行高通量表型分析，以及成年饲养的空间和技术要求。在这里，我们描述了一个实验流程，该流程将高效的 CRISPR/Cas9 诱变与功能表型筛选相结合，以鉴定成年斑马鱼脊髓修复所需的基因。我们使用 CRISPR/Cas9 双引导 RNA 蛋白，在 28 个靶位点对 17 个基因进行了选择性和组合性诱变，在成年 F0“crispants”中的效率超过 85%。我们发现毛细管电泳是测量插入缺失频率的可靠方法。使用可量化的行为测定法，我们鉴定出七个单基因或双基因 crispants 在脊髓损伤后功能恢复减少。为了排除脱靶效应，我们产生了生殖系突变，该突变重现了 crispant 再生表型。本研究提供了一个平台，将高效的体细胞诱变与功能表型读数相结合，可在成年斑马鱼中进行中等至大规模的遗传研究。
