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用于评估产生功能丧失等位基因sgRNA的评分系统
作者:小柯机器人 发布时间:2020/6/9 18:21:00

奥地利维也纳生物中心Ulrich Elling和Johannes Zuber研究小组合作取得一项新成果。经过不懈努力,他们发现多层VBC评分系统可预测有效产生功能丧失等位基因的sgRNA。 这一研究成果于2020年6月8日发表在《自然—方法学》上。

使用优化的筛选工作流程,研究人员在癌细胞系和胚胎干细胞中进行了必需基因组的筛选,并获得了无法用常用移码突变频率解释的敲出效率。研究人员发现该优异效应的大小主要取决于框内突变对蛋白质功能的影响,这可以根据氨基酸组成和保守性进行预测。

研究人员将蛋白质特征整合到“ Bioscore”中,并将其与单向-向导RNA活性和插入缺失融合在一起形成了改进的预测因子,并建立一个可捕获CRISPR-Cas9诱变中所有相关过程的评分系统。该Vienna Bioactivity CRISPR评分系统(www.vbc-score.org)优于以前的预测工具,可以选择有效产生功能丧失等位基因的sgRNA 。

研究人员表示,CRISPR-Cas9筛选已成为一种系统探究基因功能的转化方法。这种筛选方法的质量和成功率取决于功能丧失等位基因发生的频率,尤其是在广泛应用于探究必需基因阴性选择的筛选中。

附:英文原文

Title: Multilayered VBC score predicts sgRNAs that efficiently generate loss-of-function alleles

Author: Georg Michlits, Julian Jude, Matthias Hinterndorfer, Melanie de Almeida, Gintautas Vainorius, Maria Hubmann, Tobias Neumann, Alexander Schleiffer, Thomas Rainer Burkard, Michaela Fellner, Max Gijsbertsen, Anna Traunbauer, Johannes Zuber, Ulrich Elling

Issue&Volume: 2020-06-08

Abstract: CRISPR–Cas9 screens have emerged as a transformative approach to systematically probe gene functions. The quality and success of these screens depends on the frequencies of loss-of-function alleles, particularly in negative-selection screens widely applied for probing essential genes. Using optimized screening workflows, we performed essentialome screens in cancer cell lines and embryonic stem cells and achieved dropout efficiencies that could not be explained by common frameshift frequencies. We find that these superior effect sizes are mainly determined by the impact of in-frame mutations on protein function, which can be predicted based on amino acid composition and conservation. We integrate protein features into a ‘Bioscore’ and fuse it with improved predictors of single-guide RNA activity and indel formation to establish a score that captures all relevant processes in CRISPR–Cas9 mutagenesis. This Vienna Bioactivity CRISPR score (www.vbc-score.org) outperforms previous prediction tools and enables the selection of sgRNAs that effectively produce loss-of-function alleles.

DOI: 10.1038/s41592-020-0850-8

Source: https://www.nature.com/articles/s41592-020-0850-8

期刊信息

Nature Methods:《自然—方法学》,创刊于2004年。隶属于施普林格·自然出版集团,最新IF:28.467
官方网址:https://www.nature.com/nmeth/
投稿链接:https://mts-nmeth.nature.com/cgi-bin/main.plex