CRISPR/Cas Tools

This page describes a list of software platforms and bioinformatics tools built to facilitate the design of guide RNAs (gRNAs) for use with the CRISPR/Cas system.

The CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated nucleases) system was originally discovered to be an acquired immune response mechanism used by archaea and bacteria. It has since been adopted for use as a tool in the genetic engineering of higher organisms.

Designing an appropriate gRNA is an important element of genome editing with the CRISPR/Cas system. A gRNA can and at times does have unintended interactions ("off-targets") with other locations of the genome of interest. For a given candidate gRNA, these tools report its list of potential off-targets in the genome thereby allowing the designer to evaluate its suitability prior to embarking on any experiments.

Scientists have also begun exploring the mechanics of the CRISPR/Cas system and what governs how good, or active, a gRNA is at directing the Cas nuclease to a specific location of the genome of interest.[1][2] As a result of this work, new methods of assessing a gRNA for its 'activity' have been published,[1][2] and it is now best practice to consider both the unintended interactions of a gRNA as well as the predicted activity of a gRNA at the design stage.

The below table lists available tools and their attributes, and includes links to the corresponding websites.

List of CRISPR/Cas tools
Tool Name Provider Searches whole genome for targets Returns all targets of genome Seed span and location can be defined Maximum number of mismatches supported Predicts gRNA activity Available Protospacer adjacent motif (PAM) sequences Annotation is reported gRNA suggestion or scoring External Link References
Synthego Design Tool Synthego Yes (over 120,000 genomes No (Optimized for Knockout) Yes 3 Yes NGG Yes (RefSeq, Ensembl, Gencode) Yes Webserver [3]
CASTING Caagle Yes Yes No 3 No NGG and NAG No Yes Webserver [4]
Benchling CRISPR gRNA DesignBenchlingYesYesYes4 YesUser customizableYesYesWebserver-
Breaking-Cas Spanish National Center for Biotechnology Yes (over 1000 genomes) Yes Yes (by weights) 4 No User customizable Yes Yes Webserver [5]
Cas-OFFinderSeoul National UniversityYesYesNo0-10 NoNGG, NRG, NNAGAAW, NNNNGMTTNoYesWebserver Source code[6]
CRISPORUniversity of California, Santa Cruz TEFORYes (over 200 genomes)YesNo4 YesNGG, NGA, NGCG, NNAGAA, NGGNG, NNGRRT, NNNRRT, NNNNGMTT, NNNNACA, TTTNYesYesWebserver Source code[7]
CCTopUniversity of HeidelbergYesYesPartial5 (0-5) NoNGG, NRG, NNGRRT, NNNNGATT, NNAGAAW, NAAAACYesYesWebserver[8]
CHOPCHOPHarvard UniversityYesYesPartial0, 2 NoNGG, NNAGAA, NNNNGANNNoYesWebserver[9]
CHOPCHOP v2University of BergenYesYesYes3 (0-3) YesUser customizableYesYesWebserver Source code[10]
CODDayong GuoNoNoNo0, 3, 5, 8 NoNGG and NAGNoYesWebserver-
CRISPR Configurator & Specificity ToolDharmacon, Inc.Yes (over 30 species)YesYes8 (gaps or mismatches) InternallyNGG and NAGmRNA exons, Links to UCSC genome browser annotationsNoWebserver Specificity Tool-
CRISPR DesignZhang Lab, MITYesNoNo4 NoNGG and NAGmRNA exonsYesWebserver[11]
CRISPRdirectDatabase Center for Life Science (DBCLS)Yes (over 200 species)YesNoAny number NoNNNYesYesWebserver[12]
CRISPR gRNA Design ToolDNA2.0YesYesNo0-10 NoNGG, NAGGenbank annotations: Gene, misc_RNA, ncRNA, CDS, exonYesWebserver-
CRISPR LifePipeLife and SoftYesYesYes0-5yesNGG, NGA, NGCG, TTTN, NNGRRTYesYesWebserver-
CRISPRseekBioconductorYesYesNoAny number NoUser customizablemRNA exonsYesSource code[13]
DESKGENDesktop GeneticsYesYesYesAny number YesFully user customizableYesYesWebserver[14]
Genedata SelectorGenedataYesYesYesCustomizable NoCustomizableYes/Pre-loadedCustomizableWebserver-
Geneious CRISPR Site FinderGeneiousYesYesYesAny number YesUser customizableYesYesGeneious[1]
GT-Scan CSIRO & EMBL-ABR Yes Yes Yes 3 (0-3) No User customizable Links to Ensembl genome browser Yes Webserver [15]
Off-SpotterThomas Jefferson UniversityYesYesYes0-5 NGG, NAG, NNNNACA, NNGRRT (R is A or G)mRNA exons, unspliced mRNA, mRNA, 5'UTR, CDS, 3'UTR, unspliced lincRNA, lincRNAUser customizableWebserver Source code Remote batch submission[16]
sgRNA DesignerBroad InstituteNoNoNo0 YesNGGCDS (if searching by transcript ID)YesWebserver Source code[1]

References

  1. 1 2 3 4 Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, Sullender M, Ebert BL, Xavier RJ, Root DE (2014). "Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation". Nat. Biotechnol. 32 (12): 1262–7. doi:10.1038/nbt.3026. PMC 4262738. PMID 25184501.
  2. 1 2 Chari, Raj; Mali, Prashant; Moosburner, Mark; Church, George M (2015-01-01). "Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach". Nature Methods. 12 (9): 823–826. doi:10.1038/nmeth.3473. PMC 5292764. PMID 26167643.
  3. ↑ TechCrunch. "Synthego’s genetic toolkit aims to make CRISPR more accessible | May 2017" Retrieved 23 January 2018.
  4. ↑ Enkler, Ludovic; Richer, Delphine; Marchand, Anthony L.; Ferrandon, Dominique; Jossinet, Fabrice (2016-10-21). "Genome engineering in the yeast pathogen Candida glabrata using the CRISPR-Cas9 system". Scientific Reports. 6: 35766. doi:10.1038/srep35766. ISSN 2045-2322. PMC 5073330. PMID 27767081.
  5. ↑ Oliveros, Juan C.; Franch, Mònica; Tabas-Madrid, Daniel; San-León, David; Montoliu, Lluis; Cubas, Pilar; Pazos, Florencio (2016-07-08). "Breaking-Cas-interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes". Nucleic Acids Research. 44 (W1): W267–271. doi:10.1093/nar/gkw407. ISSN 1362-4962. PMC 4987939. PMID 27166368.
  6. ↑ Bae S, Park J, Kim JS (2014). "Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases". Bioinformatics. 30 (10): 1473–5. doi:10.1093/bioinformatics/btu048. PMC 4016707. PMID 24463181.
  7. ↑ Haeussler, Schoenig, Eckert, et al. (2016). "Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR". Genome Biology. 17 (1): 148. doi:10.1186/s13059-016-1012-2. PMC 4934014. PMID 27380939.
  8. ↑ Stemmer M, Thumberger T, Del Sol Keyer M, Wittbrodt J, Mateo JL (2015). "CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool". PLOS ONE. 10 (4): e0124633. doi:10.1371/journal.pone.0124633. PMC 4409221. PMID 25909470.
  9. ↑ Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E (2014). "CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing". Nucleic Acids Res. 42 (W1): W401–7. doi:10.1093/nar/gku410. PMC 4086086. PMID 24861617.
  10. ↑ Labun K, Montague TG, Gagnon JA, Thyme SB, Valen E (2016). "CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering". Nucleic Acids Res. 44 (W1): W272–6. doi:10.1093/nar/gkw398. PMC 4987937. PMID 27185894.
  11. ↑ Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F (2013). "DNA targeting specificity of RNA-guided Cas9 nucleases". Nat. Biotechnol. 31 (9): 827–32. doi:10.1038/nbt.2647. hdl:1721.1/102691. PMC 3969858. PMID 23873081.
  12. ↑ Naito Y, Hino K, Bono H, Ui-Tei K (2015). "CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites". Bioinformatics. 31 (7): 1120–3. doi:10.1093/bioinformatics/btu743. PMC 4382898. PMID 25414360.
  13. ↑ Zhu LJ, Holmes BR, Aronin N, Brodsky MH (2014). "CRISPRseek: A Bioconductor Package to Identify Target-Specific Guide RNAs for CRISPR-Cas9 Genome-Editing Systems". PLOS ONE. 9 (9): e108424. doi:10.1371/journal.pone.0108424. PMC 4172692. PMID 25247697.
  14. ↑ "Desktop Genetics Announces the Launch of DeskGen Gene Editing Platform". American Laboratory. 2015.
  15. ↑ O’Brien, Aidan; Bailey, Timothy L. (2014-09-15). "GT-Scan: identifying unique genomic targets". Bioinformatics. 30 (18): 2673–2675. doi:10.1093/bioinformatics/btu354. ISSN 1367-4803. PMC 4155256. PMID 24860161.
  16. ↑ Pliatsika V, Rigoutsos I (2015). ""Off-Spotter": very fast and exhaustive enumeration of genomic lookalikes for designing CRISPR/ Cas guide RNAs". Biol. Direct. 10 (1): 4. doi:10.1186/s13062-015-0035-z. PMC 4326336. PMID 25630343.
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