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.

CRISPR-Cas9 筛选已成为系统探测基因功能的革命性方法。这些筛选的质量和成功取决于功能丧失等位基因的频率，特别是在广泛应用于探测必需基因的负选择筛选中。使用优化的筛选工作流程，我们在癌细胞系和胚胎干细胞中进行了基本组筛选，并实现了常见移码频率无法解释的退出效率。我们发现这些优异的效应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。我们将蛋白质特征整合到“Bioscore”中，并将其与单引导 RNA 活性和插入缺失形成的改进预测因子融合，以建立一个捕获 CRISPR-Cas9 诱变中所有相关过程的评分。维也纳生物活性 CRISPR 评分 (www.vbc-score.org) 优于之前的预测工具，能够选择有效产生功能丧失等位基因的 sgRNA。