Efficient CRISPR/Cas9 mutagenesis for neurobehavioral screening in adult zebrafish.
Efficient CRISPR/Cas9 mutagenesis for neurobehavioral screening in adult zebrafish.
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DOI：
10.1093/g3journal/jkab089
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发表时间：
2021-08-07
期刊：
影响因子：
--
通讯作者：
Mokalled MH
中科院分区：
文献类型：
--
作者：
Klatt Shaw D;Mokalled MH
成年斑马鱼是研究疾病发展和组织再生机制的首选脊椎动物模型。在这一期中，Shaw和mokaled利用高效的基因组编辑工具，在17个基因中引入了28个突变，效率超过85%。一项可量化的游泳耐力测试确定了脊髓损伤后功能恢复减少的7个突变。这项研究为在成年斑马鱼身上进行中到大规模的神经行为筛选提供了一个实验管道。成年斑马鱼被广泛用于研究疾病发展和组织再生的机制。然而，在成年斑马鱼中进行大规模遗传研究的前景一直面临着许多生物学和技术上的挑战，包括成鱼组织难以获得高通量表型，以及成鱼养殖的空间和技术要求。在这里，我们描述了一个结合高效CRISPR/Cas9突变和功能表型筛选的实验管道，以鉴定成年斑马鱼脊髓修复所需的基因。利用CRISPR/Cas9双导核糖核蛋白，我们在成年F0“crispants”中展示了28个靶点上17个基因的选择性和组合诱变，效率超过85%。我们发现毛细管电泳是一种可靠的测量indel频率的方法。使用可量化的行为分析，我们确定了7个单基因或重复基因crispants在脊髓损伤后功能恢复减少。为了排除脱靶效应，我们产生了再现脆嫩再生表型的种系突变。本研究提供了一个平台，将高效体细胞诱变与功能性表型读数相结合，在成年斑马鱼中进行中到大规模的遗传研究。
Adult zebrafish are a premier vertebrate model to interrogate mechanisms of disease development and tissue regeneration. In this issue, Shaw and Mokalled used high efficiency genome editing tools to introduce 28 mutations in 17 genes with efficiencies exceeding 85%. A quantifiable swim endurance test identified 7 mutations with reduced functional recovery after spinal cord injury. This study provides an experimental pipeline to perform medium- to large-scale neurobehavioral screens in adult zebrafish. 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.
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影响因子：
25
作者：
Chen J;Poskanzer KE;Freeman MR;Monk KR
通讯作者：
Monk KR
影响因子：
3.7
作者：
Hwang WY;Fu Y;Reyon D;Maeder ML;Kaini P;Sander JD;Joung JK;Peterson RT;Yeh JR
通讯作者：
Yeh JR
影响因子：
7.7
作者：
Askary, Amjad;Smeeton, Joanna;Crump, J. Gage
通讯作者：
Crump, J. Gage
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
影响因子：
3.3
作者：
Henke, Katrin;Daane, Jacob M.;Harris, Matthew P.
通讯作者：
Harris, Matthew P.