用於評估產生功能喪失等位基因sgRNA的評分系統

用於評估產生功能喪失等位基因sgRNA的評分系統

作者：

小柯機器人

發布時間：2020/6/9 18:21:00

奧地利維也納生物中心Ulrich Elling和Johannes Zuber研究小組合作取得一項新成果。經過不懈努力，他們發現多層VBC評分系統可預測有效產生功能喪失等位基因的sgRNA。 這一研究成果於2020年6月8日發表在《自然—方法學》上。

使用優化的篩選工作流程，研究人員在癌細胞系和胚胎幹細胞中進行了必需基因組的篩選，並獲得了無法用常用移碼突變頻率解釋的敲出效率。研究人員發現該優異效應的大小主要取決於框內突變對蛋白質功能的影響，這可以根據胺基酸組成和保守性進行預測。

研究人員將蛋白質特徵整合到「 Bioscore」中，並將其與單向-嚮導RNA活性和插入缺失融合在一起形成了改進的預測因子，並建立一個可捕獲CRISPR-Cas9誘變中所有相關過程的評分系統。該Vienna Bioactivity CRISPR評分系統（www.vbc-score.org）優於以前的預測工具，可以選擇有效產生功能喪失等位基因的sgRNA 。

研究人員表示，CRISPR-Cas9篩選已成為一種系統探究基因功能的轉化方法。這種篩選方法的質量和成功率取決於功能喪失等位基因發生的頻率，尤其是在廣泛應用於探究必需基因陰性選擇的篩選中。

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