CRISPR/Cas tools

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CRISPR-Cas design tools are computer software platforms and bioinformatics tools used to facilitate the design of guide RNAs (gRNAs) for use with the CRISPR/Cas gene editing 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.

List of CRISPR-Cas design 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 References
CRISPRon, CRISPRoff Center for non-coding RNA in Technology and Health, University of Copenhagen Yes Yes Yes All Yes NGG, NGA, NAG Yes Yes [3],[4]
Invitrogen TrueDesign Genome Editor Thermo Fisher Scientific Yes Yes No 3 No NGG Yes Yes [5]
Breaking-Cas Spanish National Center for Biotechnology Yes (over 1000 genomes) Yes Yes (by weights) 4 No User customizable Yes Yes [6]
Cas-OFFinder Seoul National University Yes Yes No 0-10 No NGG, NRG, NNAGAAW, NNNNGMTT No Yes [7]
CASTING Caagle Yes Yes No 3 No NGG and NAG No Yes [8]
CRISPy Technical University of Denmark Yes Yes No All No NGG Yes Yes [9]
CCTop University of Heidelberg Yes Yes Partial 5 (0-5) Yes NGG, NRG, NNGRRT, NNNNGATT, NNAGAAW, NAAAAC Yes Yes [10]
CHOPCHOP Harvard University Yes Yes Partial 0, 2 No NGG, NNAGAA, NNNNGANN No Yes [11]
CHOPCHOP v2 University of Bergen Yes Yes Yes 3 (0-3) Yes User customizable Yes Yes [12]
CRISPOR University of California, Santa Cruz TEFOR Yes (over 200 genomes) Yes No 4 Yes NGG, NGA, NGCG, NNAGAA, NGGNG, NNGRRT, NNNRRT, NNNNGMTT, NNNNACA, TTTN Yes Yes [13]
CRISPR Design Zhang Lab, MIT Yes No No 4 No NGG and NAG mRNA exons Yes [14]
CRISPRdirect Database Center for Life Science (DBCLS) Yes (over 200 species) Yes No Any number No NNN Yes Yes [15]
CRISPRscan Giraldez Lab, Yale Yes Yes No 4 Yes NGG, TTTV, TTTN Yes Yes [16]
CRISPRseek Bioconductor Yes Yes No Any number No User customizable mRNA exons Yes [17]
DESKGEN Desktop Genetics Yes Yes Yes Any number Yes Fully user customizable Yes Yes [18]
GuideScan GuideScan Yes Yes Yes 3 on website and customizable with command line Yes NGG/NAG on website and customizable with command line Yes Yes [19]
GT-Scan CSIRO & EMBL-ABR Yes Yes Yes 3 (0-3) No User customizable Links to Ensembl genome browser Yes [20]
Off-Spotter Thomas Jefferson University Yes Yes Yes 0-5 NGG, NAG, NNNNACA, NNGRRT (R is A or G) mRNA exons, unspliced mRNA, mRNA, 5'UTR, CDS, 3'UTR, unspliced lincRNA, lincRNA User customizable [21]
sgRNA Designer Broad Institute No No No 0 Yes NGG CDS (if searching by transcript ID) Yes [1]
Synthego Design Tool Synthego Yes (over 120,000 genomes) No (Optimized for Knockout) Yes 3 Yes NGG Yes (RefSeq, Ensembl, Gencode) Yes [22]
TUSCAN CSIRO No No No 0 Yes NGG No Yes [23]
VARSCOT CSIRO Yes Yes No 0-8 Yes User customizable No Yes [24]
CRISPR Targeted Gene Designer Horizon Discovery[permanent dead link] Yes, Multiple yes yes 4 Yes NGG, NNGRRT, YTTV, other Yes Yes (21)
GuideMaker United States Department of Agriculture, Agricultural Research Service Yes, any user supplied genome Yes Yes 0-5 Yes Any PAM site and PAM orientation Yes Yes [25]


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  10. ^ 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. Bibcode:2015PLoSO..1024633S. doi:10.1371/journal.pone.0124633. PMC 4409221. PMID 25909470.
  11. ^ Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E (July 2014). "CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing". Nucleic Acids Research. 42 (Web Server issue): W401-7. doi:10.1093/nar/gku410. PMC 4086086. PMID 24861617.
  12. ^ Labun K, Montague TG, Gagnon JA, Thyme SB, Valen E (July 2016). "CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering". Nucleic Acids Research. 44 (W1): W272–6. doi:10.1093/nar/gkw398. PMC 4987937. PMID 27185894.
  13. ^ Haeussler M, Schönig K, Eckert H, Eschstruth A, Mianné J, Renaud JB, et al. (July 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.
  14. ^ Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, et al. (September 2013). "DNA targeting specificity of RNA-guided Cas9 nucleases". Nature Biotechnology. 31 (9): 827–32. doi:10.1038/nbt.2647. hdl:1721.1/102691. PMC 3969858. PMID 23873081.
  15. ^ Naito Y, Hino K, Bono H, Ui-Tei K (April 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.
  16. ^ Moreno-Mateos MA, Vejnar CE, Beaudoin JD, Fernandez JP, Mis EK, Khokha MK, Giraldez AJ (October 2015). "CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo". Nature Methods. 12 (10): 982–8. doi:10.1038/nmeth.3543. PMC 4589495. PMID 26322839.
  17. ^ 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. Bibcode:2014PLoSO...9j8424Z. doi:10.1371/journal.pone.0108424. PMC 4172692. PMID 25247697.
  18. ^ "Desktop Genetics Announces the Launch of DeskGen Gene Editing Platform". American Laboratory. 2015.
  19. ^ Perez AR, Pritykin Y, Vidigal JA, Chhangawala S, Zamparo L, Leslie CS, Ventura A (April 2017). "GuideScan software for improved single and paired CRISPR guide RNA design". Nature Biotechnology. 35 (4): 347–349. doi:10.1038/nbt.3804. PMC 5607865. PMID 28263296.
  20. ^ O'Brien A, Bailey TL (September 2014). "GT-Scan: identifying unique genomic targets". Bioinformatics. 30 (18): 2673–5. doi:10.1093/bioinformatics/btu354. PMC 4155256. PMID 24860161.
  21. ^ Pliatsika V, Rigoutsos I (January 2015). ""Off-Spotter": very fast and exhaustive enumeration of genomic lookalikes for designing CRISPR/Cas guide RNAs". Biology Direct. 10 (1): 4. doi:10.1186/s13062-015-0035-z. PMC 4326336. PMID 25630343.
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  23. ^ Wilson LO, Reti D, O'Brien AR, Dunne RA, Bauer DC (April 2018). "High Activity Target-Site Identification Using Phenotypic Independent CRISPR-Cas9 Core Functionality". The CRISPR Journal. 1 (2): 182–190. doi:10.1089/crispr.2017.0021. PMID 31021206.
  24. ^ Wilson LO, Hetzel S, Pockrandt C, Reinert K, Bauer DC (June 2019). "VARSCOT: variant-aware detection and scoring enables sensitive and personalized off-target detection for CRISPR-Cas9". BMC Biotechnology. 19 (1): 40. doi:10.1186/s12896-019-0535-5. PMC 6598273. PMID 31248401.
  25. ^ Poudel R, Rodriguez LT, Reisch CR, Rivers AR (April 2022). "GuideMaker: Software to design CRISPR-Cas guide RNA pools in non-model genomes". GigaScience. 11. doi:10.1093/gigascience/giac007. PMID 35365834. S2CID 235700000.