Choosing the Right Tool for Designing Guide RNAs
The first step of CRISPR/Cas9 gene editing is designing a single guide RNA (sgRNA) to target your gene of interest. Because sgRNAs are solely responsible for recruiting Cas9 to specific genomic loci, optimal sgRNA design is critical for successful gene-editing experiments. There are many web-based tools available for sgRNA design, each of which has different features and advantages. The information provided here will help you choose the best tool for your specific research objective.
Several web-based tools available
Web-based sgRNA design tools typically require that users input a DNA sequence, genomic location or gene name for each gene of interest, and indicate a species. An algorithm specific to each tool outputs a list of candidate guide sequences with corresponding predicted off-target sites for each input (Wu et al., 2014). Most tools aim to provide guide sequences that minimize the likelihood of off-target effects, but the methods they employ vary. For example, the CRISPR Design Tool (Hsu et al., 2013) uses empirical data from previous mutagenic studies. Alternatively, CasFinder (Aach et al., 2014) and E-CRISP (Heigwer et al., 2014) incorporate specific user-defined penalties based on the number and position of mismatches relative to the guide sequence in order to rank the potential for off-target effects.
Tools for specific applications
Some sgRNA design tools have been developed for specific applications. CRISPR-ERA (Liu et al., 2015) is the only currently available tool that designs sgRNAs specifically for gene repression or activation, while FlyCRISPR (Gratz et al., 2013) focuses on applications in fly, beetle, and worm species, including the popular model organisms Drosophila melanogaster and Caenorhabditis elegans. Presently, the design tool featured on the Benchling website is the only one that can generate candidate sgRNAs that are compatible with alternative nucleases such as Staphylococcus aureus Cas9 (Ran et al., 2015) and Cpf1 (Zetsche et al., 2015). Given the uniqueness of each tool, we recommend that you use multiple approaches during the sgRNA design process and choose guide sequences that are consistently predicted to perform well.
A selection of freely available tools
The table below provides a list of web-based tools for sgRNA design. For simplicity, we have only included those that are free and don’t require a subscription. For each tool, we have indicated whether there is a convenient graphical user interface or if the user has to download a script. If you would prefer to design sgRNAs manually, please visit Designing Guide RNAs to learn more.
|Noteworthy Features of Web-Based sgRNA Design Tools|
|Name||Graphical user interface||Available species||Input||Output||Ranked list|
|CRISPR Design Tool||Yes||15||DNA sequence||Candidate guide sequences and off-target loci||Yes|
|CHOPCHOP||Yes||23||DNA sequence, gene name, genomic location||Candidate guide sequences and off-target loci||No|
|CasFinder||No (Perl script)||User input||DNA sequence||Candidate guide sequences and off-target loci||Yes|
|Cas-OFFFinder||Yes||11||Guide sequence||Off-target loci for guide sequences||No|
|FlyCRISPR||Yes||18||DNA sequence||Candidate guide sequences and off-target loci||No|
|E-CRISP||Yes||31||DNA sequence or gene name||Candidate guide sequences and off-target loci||Yes|
Guide RNA Sequence Design
|Yes||10||DNA sequence||Candidate guide sequences and off-target loci||No|
|CasOT||No (Perl script)||User input||Guide sequence||Off-target loci and additional guide sequences||No|
|CRISPR-ERA||Yes||9||DNA sequence, gene name, or TSS location||Candidate guide sequences and distances to TSS||Yes|
|Benchling||Yes||5||DNA sequence or gene name||Candidate guide sequences and off-target loci||Yes|
- Aach, et al. (2014) Flexible algorithm for identifying specific Cas9 targets in genomes. BioRxiv, Cold Spring Harbor Labs. doi: http://dx.doi.org/10.1101/005074.
- Bae, et al. (2014) Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 30(10):1473–1475.
- Gratz, et al. (2014) Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics. 196(4):961–971.
- Heigwer, et al. (2014) E-CRISP: fast CRISPR target site identification. Nat Methods. 11(2):122–123.
- Hsu, et al. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 31(9):827–832.
- Ma, et al. (2013) A guide RNA sequence design platform for the CRISPR/Cas9 system for model organism genomes. Biomed Res Int. doi:http://doi.org/10.1155/2013/270805.
- Montague, et al. (2014) CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42(W1):W401–W407.
- Liu, et al. (2015) CRISPR-ERA: a comprehensive design tool for CRISPR-mediated gene editing, repression and activation. Bioinformatics. 31(22):3676–3678.
- Ran, et al. (2015) In vivo genome editing using Staphylococcus aureus Cas9. Nature. 520(7546):186–191.
- Wu, et al. (2014) Target specificity of the CRISPR-Cas9 system. Quant Biol. 2(2):59–70.
- Xiao, et al. (2014) CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics. 30(8):1180–1182.
- Zetsche, et al. (2015) Cpf1 is a single RNA-guided endonuclease of a Class 2 CRISPR-Cas System. Cell. 163(3):759–771.