Genes & Human Disease Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, 73104, USA.
Sci Rep. 2020 Feb 21;10(1):3172. doi: 10.1038/s41598-020-59986-1.
Powerful and simple, RNA-guided CRISPR/Cas9 technology is a versatile genome editing tool that has revolutionized targeted mutagenesis. CRISPR-based genome editing has enabled large-scale functional genetic studies through the generation of gene knockouts in a variety of model organisms including zebrafish, and can be used to target multiple genes simultaneously. One of the challenges associated with the large scale application of this technique to zebrafish is the lack of a cost-effective method by which to identify mutants. To address this, we optimized the high-throughput, high-resolution fluorescent PCR-based fragment analysis method to develop MultiFRAGing - a robust and cost-effective method to genotype multiple targets in a single reaction. Our approach can identify indels in up to four targets from a single reaction, which represents a four-fold increase in genotyping throughput. This method can be used by any laboratory with access to capillary electrophoresis-based sequencing equipment.
强大而简单的 RNA 引导的 CRISPR/Cas9 技术是一种通用的基因组编辑工具，它彻底改变了靶向诱变。基于 CRISPR 的基因组编辑通过在包括斑马鱼在内的各种模式生物中生成基因敲除，实现了大规模的功能遗传研究，并且可以同时靶向多个基因。将这项技术大规模应用于斑马鱼的挑战之一是缺乏一种经济有效的方法来鉴定突变体。为了解决这个问题，我们优化了高通量、高分辨率的荧光 PCR 片段分析方法，开发了 MultiFRAGing-一种在单个反应中同时对多个靶标进行基因分型的稳健且经济有效的方法。我们的方法可以从单个反应中鉴定多达四个靶标的插入缺失，这代表基因分型通量提高了四倍。任何有毛细管电泳测序设备的实验室都可以使用这种方法。
