比较使用 CRISPR-RfxCas13d 在斑马鱼胚胎中进行高通量 RNA 沉默的机器人和人工注射方法。

IF 2.2 4区 工程技术 Q3 BIOCHEMICAL RESEARCH METHODS
BioTechniques Pub Date : 2024-05-01 Epub Date: 2024-02-29 DOI:10.2144/btn-2023-0062
Joaquin Abugattas-Nuñez Del Prado, Yi Ding, Jan de Sonneville, Kees-Jan van der Kolk, Miguel A Moreno-Mateos, Edward Málaga-Trillo, Herman P Spaink
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引用次数: 0

摘要

在这项研究中,作者比较了CRISPR-RfxCas13d(CasRx)系统在斑马鱼胚胎中进行mRNA敲除和Cas9介导的DNA靶向的自动机器人和人工注射方法的效率。他们以无尾(TBXTA)基因为靶标,评估了诱导的胚胎表型,并进行了原理验证。Cas9 和 CasRx 系统都导致了 TBXTA 的功能缺失表型。与 mRNA 相比,Cas9 蛋白表现出更高比例的严重表型,而 CasRx 蛋白和 mRNA 表现出相似的效率。机器人注射和人工注射的表型比例和死亡率相当。研究结果凸显了 RNA 靶向 CRISPR 效应器在精确基因敲除方面的潜力,并支持以每个胚胎 1.0 秒的速度进行自动显微注射,作为人工方法的高通量替代方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Comparing robotic and manual injection methods in zebrafish embryos for high-throughput RNA silencing using CRISPR-RfxCas13d.

In this study, the authors compared the efficiency of automated robotic and manual injection methods for the CRISPR-RfxCas13d (CasRx) system for mRNA knockdown and Cas9-mediated DNA targeting in zebrafish embryos. They targeted the no tail (TBXTA) gene as a proof-of-principle, evaluating the induced embryonic phenotypes. Both Cas9 and CasRx systems caused loss of function phenotypes for TBXTA. Cas9 protein exhibited a higher percentage of severe phenotypes compared with mRNA, while CasRx protein and mRNA showed similar efficiency. Both robotic and manual injections demonstrated comparable phenotype percentages and mortality rates. The findings highlight the potential of RNA-targeting CRISPR effectors for precise gene knockdown and endorse automated microinjection at a speed of 1.0 s per embryo as a high-throughput alternative to manual methods.

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来源期刊
BioTechniques
BioTechniques 工程技术-生化研究方法
CiteScore
4.40
自引率
0.00%
发文量
68
审稿时长
3.3 months
期刊介绍: BioTechniques is a peer-reviewed, open-access journal dedicated to publishing original laboratory methods, related technical and software tools, and methods-oriented review articles that are of broad interest to professional life scientists, as well as to scientists from other disciplines (e.g., chemistry, physics, computer science, plant and agricultural science and climate science) interested in life science applications for their technologies. Since 1983, BioTechniques has been a leading peer-reviewed journal for methods-related research. The journal considers: Reports describing innovative new methods, platforms and software, substantive modifications to existing methods, or innovative applications of existing methods, techniques & tools to new models or scientific questions Descriptions of technical tools that facilitate the design or performance of experiments or data analysis, such as software and simple laboratory devices Surveys of technical approaches related to broad fields of research Reviews discussing advancements in techniques and methods related to broad fields of research Letters to the Editor and Expert Opinions highlighting interesting observations or cautionary tales concerning experimental design, methodology or analysis.
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