Brocal Isabel, White Richard J, Dooley Christopher M, Carruthers Samantha N, Clark Richard, Hall Amanda, Busch-Nentwich Elisabeth M, Stemple Derek L, Kettleborough Ross N W
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
Twist Bioscience, 455 Mission Bay Boulevard South, San Francisco, CA, 94158, United States.
BMC Genomics. 2016 Mar 24;17:259. doi: 10.1186/s12864-016-2563-z.
The CRISPR/Cas9 system is a prokaryotic immune system that infers resistance to foreign genetic material and is a sort of 'adaptive immunity'. It has been adapted to enable high throughput genome editing and has revolutionised the generation of targeted mutations.
We have developed a scalable analysis pipeline to identify CRISPR/Cas9 induced mutations in hundreds of samples using next generation sequencing (NGS) of amplicons. We have used this system to investigate the best way to screen mosaic Zebrafish founder individuals for germline transmission of induced mutations. Screening sperm samples from potential founders provides much better information on germline transmission rates and crucially the sequence of the particular insertions/deletions (indels) that will be transmitted. This enables us to combine screening with archiving to create a library of cryopreserved samples carrying known mutations. It also allows us to design efficient genotyping assays, making identifying F1 carriers straightforward.
The methods described will streamline the production of large numbers of knockout alleles in selected genes for phenotypic analysis, complementing existing efforts using random mutagenesis.
CRISPR/Cas9系统是一种原核免疫系统，可抵御外来遗传物质，属于一种“适应性免疫”。它已被改造用于实现高通量基因组编辑，并彻底改变了靶向突变的产生方式。
我们开发了一种可扩展的分析流程，通过对扩增子进行下一代测序（NGS）来识别数百个样本中CRISPR/Cas9诱导的突变。我们使用该系统研究了筛选嵌合斑马鱼奠基个体以实现诱导突变种系传递的最佳方法。筛选潜在奠基个体的精子样本能提供关于种系传递率的更好信息，关键是能提供将被传递的特定插入/缺失（indel）序列。这使我们能够将筛选与存档相结合，创建一个携带已知突变的冷冻保存样本库。它还使我们能够设计高效的基因分型检测方法，从而直接鉴定F1携带者。
所描述的方法将简化为进行表型分析而在选定基因中产生大量敲除等位基因的过程，对使用随机诱变的现有工作起到补充作用。
