Cao Qingyi, Ma Jian, Chen Chen-Hao, Xu Han, Chen Zhi, Li Wei, Liu X Shirley
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang, P. R. China.
Department of Bioinformatics, School of Life Science and Technology, Tongji University, Shanghai, P. R. China.
PLoS One. 2017 Sep 5;12(9):e0184281. doi: 10.1371/journal.pone.0184281. eCollection 2017.
The recently developed CRISPR screen technology, based on the CRISPR/Cas9 genome editing system, enables genome-wide interrogation of gene functions in an efficient and cost-effective manner. Although many computational algorithms and web servers have been developed to design single-guide RNAs (sgRNAs) with high specificity and efficiency, algorithms specifically designed for conducting CRISPR screens are still lacking. Here we present CRISPR-FOCUS, a web-based platform to search and prioritize sgRNAs for CRISPR screen experiments. With official gene symbols or RefSeq IDs as the only mandatory input, CRISPR-FOCUS filters and prioritizes sgRNAs based on multiple criteria, including efficiency, specificity, sequence conservation, isoform structure, as well as genomic variations including Single Nucleotide Polymorphisms and cancer somatic mutations. CRISPR-FOCUS also provides pre-defined positive and negative control sgRNAs, as well as other necessary sequences in the construct (e.g., U6 promoters to drive sgRNA transcription and RNA scaffolds of the CRISPR/Cas9). These features allow users to synthesize oligonucleotides directly based on the output of CRISPR-FOCUS. Overall, CRISPR-FOCUS provides a rational and high-throughput approach for sgRNA library design that enables users to efficiently conduct a focused screen experiment targeting up to thousands of genes. (CRISPR-FOCUS is freely available at http://cistrome.org/crispr-focus/).
最近开发的基于CRISPR/Cas9基因组编辑系统的CRISPR筛选技术,能够以高效且经济高效的方式对基因功能进行全基因组检测。尽管已经开发了许多计算算法和网络服务器来设计具有高特异性和效率的单向导RNA(sgRNA),但专门为进行CRISPR筛选设计的算法仍然缺乏。在此,我们展示了CRISPR-FOCUS,这是一个基于网络的平台,用于搜索和优先选择用于CRISPR筛选实验的sgRNA。以官方基因符号或RefSeq ID作为唯一必需的输入,CRISPR-FOCUS基于多个标准对sgRNA进行筛选和排序,包括效率、特异性、序列保守性、异构体结构,以及包括单核苷酸多态性和癌症体细胞突变在内的基因组变异。CRISPR-FOCUS还提供预定义的阳性和阴性对照sgRNA,以及构建体中的其他必要序列(例如,驱动sgRNA转录的U6启动子和CRISPR/Cas9的RNA支架)。这些功能允许用户直接根据CRISPR-FOCUS的输出合成寡核苷酸。总体而言,CRISPR-FOCUS为sgRNA文库设计提供了一种合理且高通量的方法,使用户能够高效地进行针对多达数千个基因的聚焦筛选实验。(可在http://cistrome.org/crispr-focus/免费获取CRISPR-FOCUS)