Rapid Generation of Pigment Free, Immobile Zebrafish Embryos and Larvae in Any Genetic Background Using CRISPR-Cas9 dgRNPs.

IF 1.4 4区生物学 Q4 DEVELOPMENTAL BIOLOGY

Zebrafish Pub Date : 2021-08-01 Epub Date: 2021-06-02 DOI:10.1089/zeb.2021.0011

Andrew E Davis, Daniel Castranova, Brant M Weinstein

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引用次数: 4

Abstract

The ability to carry out high-resolution, high-magnification optical imaging of living animals is one of the most attractive features of the zebrafish as a model organism. However, increasing amounts of pigmentation as development proceeds and difficulties in maintaining sustained immobilization of healthy, living animals remain challenges for live imaging. Chemical treatments can be used to suppress pigment formation and movement, but these treatments can lead to developmental defects. Genetic mutants can also be used to eliminate pigment formation and immobilize animals, but maintaining these mutants in lines carrying other combinations of transgenes and mutants is difficult and laborious. In this study, we show that CRISPR duplex guide ribonucleoproteins (dgRNPs) targeting the slc45a2 (albino) and chrna1 (nic1) genes can be used to efficiently suppress pigment formation in and immobilize F0 injected animals. CRISPR dgRNPs can be used to generate pigment-free, immobile zebrafish embryos and larvae in any transgenic and/or mutant-carrying background, greatly facilitating high-resolution imaging and analysis of the many transgenic and mutant lines available in the zebrafish.

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利用CRISPR-Cas9 dgRNPs快速生成任何遗传背景下无色素、不动的斑马鱼胚胎和幼虫

对活体动物进行高分辨率、高倍率光学成像的能力是斑马鱼作为模式生物最吸引人的特征之一。然而，随着发育的进行，色素沉着量的增加以及维持健康活体动物持续固定的困难仍然是活体成像的挑战。化学处理可以抑制色素的形成和运动，但这些处理可能导致发育缺陷。基因突变体也可用于消除色素形成和固定动物，但在携带其他转基因和突变体组合的品种中维持这些突变体是困难和费力的。在本研究中，我们发现靶向slc45a2(白化病)和chrna1 (nic1)基因的CRISPR双导核糖核蛋白(dgRNPs)可有效抑制F0注射动物的色素形成并使其固定化。CRISPR dgRNPs可用于在任何转基因和/或携带突变体的背景下产生无色素、不动的斑马鱼胚胎和幼虫，极大地促进了对斑马鱼中许多转基因和突变系的高分辨率成像和分析。

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来源期刊

Zebrafish DEVELOPMENTAL BIOLOGY-ZOOLOGY

CiteScore

3.60

自引率

5.00%

发文量

审稿时长

3 months

期刊介绍： Zebrafish is the only peer-reviewed journal dedicated to the central role of zebrafish and other aquarium species as models for the study of vertebrate development, evolution, toxicology, and human disease. Due to its prolific reproduction and the external development of the transparent embryo, the zebrafish is a prime model for genetic and developmental studies. While genetically more distant from humans, the vertebrate zebrafish nevertheless has comparable organs and tissues, such as heart, kidney, pancreas, bones, and cartilage. Zebrafish introduced the new section TechnoFish, which highlights these innovations for the general zebrafish community. TechnoFish features two types of articles: TechnoFish Previews: Important, generally useful technical advances or valuable transgenic lines TechnoFish Methods: Brief descriptions of new methods, reagents, or transgenic lines that will be of widespread use in the zebrafish community Zebrafish coverage includes: Comparative genomics and evolution Molecular/cellular mechanisms of cell growth Genetic analysis of embryogenesis and disease Toxicological and infectious disease models Models for neurological disorders and aging New methods, tools, and experimental approaches Zebrafish also includes research with other aquarium species such as medaka, Fugu, and Xiphophorus.