Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR.
Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR.
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DOI：
10.1186/s13059-016-1012-2
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发表时间：
2016-07-05
期刊：
影响因子：
12.3
通讯作者：
Concordet JP
中科院分区：
文献类型：
--
作者：
Haeussler M;Schönig K;Eckert H;Eschstruth A;Mianné J;Renaud JB;Schneider-Maunoury S;Shkumatava A;Teboul L;Kent J;Joly JS;Concordet JP
CRISPR/Cas9基因组编辑技术的成功取决于指导RNA序列的选择，这是由各种网站促成的。尽管这些算法很重要且很受欢迎，但尚不清楚它们的预测与实际测量结果在多大程度上一致。我们对CRISPR/Cas9预测进行了首次独立评估。为此，我们收集了八项SpCas 9脱靶研究的数据，并将其与流行算法预测的位点进行比较。我们在一个实施中发现了问题，但发现基于序列的脱靶预测非常可靠，识别出突变率上级优于0.1%的大多数脱靶，而假阳性的数量可以大大减少脱靶分数的截止值。我们还评估了针对可用数据集的目标效率预测算法。预测和指导活动之间的相关性变化很大，特别是对斑马鱼。结合我们实验室的新数据，我们发现最佳的靶向效率预测模型在很大程度上取决于指导RNA是从U6启动子表达还是在体外转录。我们进一步证明，最好的预测可以显着减少引导筛选所花费的时间。为了让任何计划CRISPR基因组编辑实验的人都能轻松访问这些指导原则，我们建立了一个新网站（http：crispor.org），该网站可以预测脱靶，并帮助使用不同的Cas9蛋白和本文评估的八种效率评分系统为120多个基因组选择和克隆有效的指导序列。本文的在线版本（doi：10.1186/s13059-016-1012-2）包含补充材料，可供授权用户使用。
The success of the CRISPR/Cas9 genome editing technique depends on the choice of the guide RNA sequence, which is facilitated by various websites. Despite the importance and popularity of these algorithms, it is unclear to which extent their predictions are in agreement with actual measurements. We conduct the first independent evaluation of CRISPR/Cas9 predictions. To this end, we collect data from eight SpCas9 off-target studies and compare them with the sites predicted by popular algorithms. We identify problems in one implementation but found that sequence-based off-target predictions are very reliable, identifying most off-targets with mutation rates superior to 0.1 %, while the number of false positives can be largely reduced with a cutoff on the off-target score. We also evaluate on-target efficiency prediction algorithms against available datasets. The correlation between the predictions and the guide activity varied considerably, especially for zebrafish. Together with novel data from our labs, we find that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro. We further demonstrate that the best predictions can significantly reduce the time spent on guide screening. To make these guidelines easily accessible to anyone planning a CRISPR genome editing experiment, we built a new website (http://crispor.org) that predicts off-targets and helps select and clone efficient guide sequences for more than 120 genomes using different Cas9 proteins and the eight efficiency scoring systems evaluated here. The online version of this article (doi:10.1186/s13059-016-1012-2) contains supplementary material, which is available to authorized users.