Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti.
Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti.
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DOI：
10.3791/63008
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发表时间：
2021-09-10
影响因子：
1.2
通讯作者：
Adelman, Zach N.
中科院分区：
文献类型：
--
作者：
Kojin, Bianca B.;Tsujimoto, Hitoshi;Jakes, Emma;O'Leary, Sarah;Adelman, Zach N.
随着TALEN、ZFN和归巢核酸内切酶等系统的建立，苔藓基因编辑在几个实验室中已成为常规。最近，CRISPR/Cas9技术为精确基因组工程提供了一种更简单、更便宜的替代方案。在核酸酶作用后，DNA修复途径将修复断裂的DNA末端，通常引入插入缺失。这些框架外突变然后用于理解靶生物体中的基因功能。然而，一个缺点是突变个体不携带显性标记，使得突变等位基因的鉴定和追踪具有挑战性，特别是在许多实验所需的规模下。高分辨率解链分析（HRMA）是鉴定核酸序列中的变异的简单方法，并且利用PCR解链曲线来检测此类变异。这种PCR后分析方法使用具有温度斜坡控制数据捕获能力的仪器实现荧光双链DNA结合染料，并且易于扩展到96孔板格式。在这里，我们描述了一个简单的工作流程，使用HRMA快速检测CRISPR/Cas9诱导的indel，并在蚊子Ae中建立突变系。埃及人。重要的是，所有步骤都可以用少量的腿部组织进行，并且不需要牺牲生物体，允许在基因分型后进行遗传杂交或表型分析。本文详细介绍了一种快速鉴定由CRISPR/Cas9诱导的插入缺失和选择蚊子Ae中突变系的方案。使用高分辨率熔体分析。
Mosquito gene editing has become routine in several laboratories with the establishment of systems like TALENs, ZFNs, and homing endonucleases. More recently, CRISPR/Cas9 technology has offered an easier and cheaper alternative for precision genome engineering. Following nuclease action, DNA repair pathways will fix the broken DNA ends, often introducing indels. These out-of-frame mutations are then used for understanding gene function in the target organisms. A drawback, however, is that mutant individuals carry no dominant marker, making identification and tracking of mutant alleles challenging, especially at scales needed for many experiments. High-resolution melt analysis (HRMA) is a simple method to identify variations in nucleic acid sequences and utilizes PCR melting curves to detect such variations. This post-PCR analysis method implements fluorescent double-stranded DNA-binding dyes with instrumentation that has temperature ramp control data capture capability and is easily scaled to 96-well plate formats. Here we describe a simple workflow using HRMA for the rapid detection of CRISPR/Cas9-induced indels and the establishment of mutant lines in the mosquito Ae. aegypti. Critically, all steps can be performed with a small amount of leg tissue and do not require sacrificing the organism, allowing genetic crosses or phenotyping assays to be performed after genotyping. This article details a protocol for rapid identification of indels induced by CRISPR/Cas9 and selection of mutant lines in the mosquito Ae. aegypti using high resolution melt analysis.
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影响因子：
2.5
作者：
Parant, John M.;George, Stephen A.;Pryor, Rob;Wittwer, Carl T.;Yost, H. Joseph
通讯作者：
Yost, H. Joseph
影响因子：
5.6
作者：
Słomka M;Sobalska-Kwapis M;Wachulec M;Bartosz G;Strapagiel D
通讯作者：
Strapagiel D
影响因子：
3
作者：
Kojin BB;Martin-Martin I;Araújo HRC;Bonilla B;Molina-Cruz A;Calvo E;Capurro ML;Adelman ZN
通讯作者：
Adelman ZN
影响因子：
4
作者：
Tsujimoto H;Anderson MAE;Myles KM;Adelman ZN
通讯作者：
Adelman ZN
影响因子：
5.8
作者：
Larkin, M. A.;Blackshields, G.;Higgins, D. G.
通讯作者：
Higgins, D. G.