National Research Council Canada, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada.
Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK, S7N 0X2, Canada.
BMC Plant Biol. 2019 Nov 6;19(1):474. doi: 10.1186/s12870-019-2097-z.
CRISPR/Cas9 gene editing has become a revolutionary technique for crop improvement as it can facilitate fast and efficient genetic changes without the retention of transgene components in the final plant line. Lack of robust bioinformatics tools to facilitate the design of highly specific functional guide RNAs (gRNAs) and prediction of off-target sites in wheat is currently an obstacle to effective application of CRISPR technology to wheat improvement.
We have developed a web-based bioinformatics tool to design specific gRNAs for genome editing and transcriptional regulation of gene expression in wheat. A collaborative study between the Broad Institute and Microsoft Research used large-scale empirical evidence to devise algorithms (Doech et al., 2016, Nature Biotechnology 34, 184-191) for predicting the on-target activity and off-target potential of CRISPR/SpCas9 (Streptococcus pyogenes Cas9). We applied these prediction models to determine on-target specificity and potential off-target activity for individual gRNAs targeting specific loci in the wheat genome. The genome-wide gRNA mappings and the corresponding Doench scores predictive of the on-target and off-target activities were used to create a gRNA database which was used as a data source for the web application termed WheatCRISPR.
The WheatCRISPR tool allows researchers to browse all possible gRNAs targeting a gene or sequence of interest and select effective gRNAs based on their predicted high on-target and low off-target activity scores, as well as other characteristics such as position within the targeted gene. It is publicly available at https://crispr.bioinfo.nrc.ca/WheatCrispr/ .
CRISPR/Cas9 基因编辑技术已成为作物改良的一项革命性技术，因为它可以在不保留转基因成分的情况下快速有效地进行遗传改变。目前，缺乏强大的生物信息学工具来辅助设计高度特异性的功能向导 RNA（gRNA）并预测小麦中的脱靶位点，这是限制 CRISPR 技术在小麦改良中有效应用的一个障碍。
我们开发了一个基于网络的生物信息学工具，用于设计特定的 gRNA，以进行基因组编辑和小麦基因表达的转录调控。Broad 研究所和微软研究院之间的合作研究利用大规模的经验证据设计了算法（Doech 等人，2016 年，《自然生物技术》34 卷，184-191），用于预测 CRISPR/SpCas9（酿脓链球菌 Cas9）的靶活性和脱靶潜力。我们应用这些预测模型来确定针对小麦基因组中特定基因座的特定 gRNA 的靶特异性和潜在脱靶活性。全基因组 gRNA 图谱和预测靶活性和脱靶活性的相应 Doench 评分用于创建一个 gRNA 数据库，该数据库被用作称为 WheatCRISPR 的网络应用程序的数据源。
WheatCRISPR 工具允许研究人员浏览针对感兴趣的基因或序列的所有可能的 gRNA，并根据其预测的高靶活性和低脱靶活性评分以及其他特征（如目标基因内的位置）选择有效的 gRNA。它可在 https://crispr.bioinfo.nrc.ca/WheatCrispr/ 上公开获取。
