Large-scale F0 CRISPR screens in vivo using MIC-Drop.
Large-scale F0 CRISPR screens in vivo using MIC-Drop.
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DOI：
10.1038/s41596-023-00821-y
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发表时间：
2023-06
期刊：
影响因子：
14.8
通讯作者：
Peterson, Randall T. T.
中科院分区：
文献类型：
--
作者：
Parvez, Saba;Brandt, Zachary J. J.;Peterson, Randall T. T.
斑马鱼是研究动物发育、模拟遗传疾病和大规模活体功能遗传学的强大模型系统。由于CRISPR-Cas9的易用性和在靶向基因干扰方面的高效，CRISPR-Cas9最近已成为斑马鱼遗传操作的首选工具。然而，扩大高通量体内功能遗传学的技术一直是一个挑战。我们最近开发了一种名为多重混合CRISPR液滴(MIC-Drop)的方法，使大规模筛选斑马鱼CRISPR成为可能。在这里，我们概述了使用MIC-Drop在斑马鱼中进行功能遗传筛选的逐步方案。MICROP-DROP使用多路复用单引导RNA(SgRNAs)在注射的斑马鱼胚胎中产生双等位基因突变，从而允许在F0动物中进行遗传筛选。结合微流控和DNA条形码，可以从一根注射针同时定位数十到数百个基因，同时还可以追溯和快速识别导致观察到的表型的基因。MIC-Drop的主要目标受众是发育生物学家、斑马鱼遗传学家，以及对在脊椎动物模型系统中进行体内功能遗传筛选感兴趣的研究人员。微滴技术在化学生物学家手中也将被证明是有用的，他们试图识别导致斑马鱼表型变化的小分子靶标。使用MIC-Drop，一个用户可以在2-3周内进行100个基因的典型筛选。
The zebrafish is a powerful model system for studying animal development, modeling genetic diseases, and for large-scale in vivo functional genetics. Because of its ease of use and its high efficiency in targeted gene perturbation, CRISPR-Cas9 has recently gained prominence as the tool-of-choice for genetic manipulation in zebrafish. However, scaling up the technique for high-throughput in vivo functional genetics has been a challenge. We recently developed a method, Multiplexed Intermixed CRISPR Droplets (MIC-Drop), that makes large-scale CRISPR screening in zebrafish possible. Here we outline the step-by-step protocol for performing functional genetic screens in zebrafish using MIC-Drop. MIC-Drop uses multiplexed single-guide RNAs (sgRNAs) to generate biallelic mutations in injected zebrafish embryos, allowing genetic screens to be performed in F0 animals. Combining microfluidics and DNA barcoding enables simultaneous targeting of tens to hundreds of genes from a single injection needle, while also enabling retrospective and rapid identification of the genotype responsible for an observed phenotype. The primary target audiences for MIC-Drop are developmental biologists, zebrafish geneticists, and researchers interested in performing in vivo functional genetic screens in a vertebrate model system. MIC-Drop will also prove useful in the hands of chemical biologists seeking to identify targets of small molecules that cause phenotypic changes in zebrafish. Using MIC-Drop, a typical screen of 100 genes can be conducted within 2–3 weeks by a single user.
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影响因子：
14.8
作者：
Feldman, David;Funk, Luke;Le, Anna;Carlson, Rebecca J.;Leiken, Michael D.;Tsai, FuNien;Soong, Brian;Singh, Avtar;Blainey, Paul C.
通讯作者：
Blainey, Paul C.
影响因子：
25
作者：
Bahl, Armin;Engert, Florian
通讯作者：
Engert, Florian
DOI：
10.1038/s43586-021-00093-4
发表时间：
2022-02-10
期刊：
NATURE REVIEWS METHODS PRIMERS
影响因子：
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作者：
Bock, Christoph;Datlinger, Paul;Zhuang, Xiaowei
通讯作者：
Zhuang, Xiaowei
影响因子：
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作者：
Ghanta KS;Ishidate T;Mello CC
通讯作者：
Mello CC
影响因子：
3.7
作者：
Foley, Jonathan E.;yeh, Jing-Ruey J.;Maeder, Morgan L.;Reyon, Deepak;Sander, Jeffry D.;Peterson, Randall T.;Joung, J. Keith
通讯作者：
Joung, J. Keith