WheatCRISPR: a web-based guide RNA design tool for CRISPR/Cas9-mediated genome editing in wheat

Dustin Cram,Sateesh Kagale,Pankaj Bhowmik,Andrew G Sharpe,Kevin Rozwadowski,Isobel A P Parkin,Nandhakishore Rajagopalan,Miles Buchwaldt,Manoj Kulkarni

doi:10.1186/s12870-019-2097-z

Abstract

BackgroundCRISPR/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.DescriptionWe 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.ConclusionThe 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/.

Highlights

CRISPR/CRISPR-associated endonuclease (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
The WheatCRISPR tool allows researchers to browse all possible guide RNA (gRNA) 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
The Doench cutting frequency determination (CFD) algorithm was applied to the Utility and discussion WheatCRISPR provides a convenient interface to browse the gRNA database, and allows researchers to view a set of predicted gRNAs targeting a gene or sequence of interest, and select them based on their predicted on-target and off-target activity, and the position of the gRNA within the targeted gene

Summary

Conclusion

As an elegant alternative to reliance on natural or induced mutagenesis, CRISPR/Cas9-based gene editing technology has the potential to change the pace and course of crop breeding. To facilitate the application of this innovative technology in wheat, we have developed a robust web-based bioinformatics tool (WheatCRISPR) to enable selection of specific gRNAs for user-specified target gene or sequence and prediction of potential offtarget sites. In wheat there will be a few genes for which finding unique gRNAs would be difficult due to polyploidy, high content of repetitive DNA content and genes typically existing as members of multi-gene families with high levels of sequence identity. In such cases, the users may have to consider other strategies (for example, dual gRNAs) to improve targeting specificity. Availability and requirements The WheatCRISPR web application is publicly available at https://crispr.bioinfo.nrc.ca/WheatCrispr/

Findings

Background