CRISPR/Cas Tools

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CRISPR-Cas design tools are 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 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 External Link References
Benchling CRISPR gRNA Design Benchling Yes Yes Yes 4 Yes User customizable Yes Yes Webserver -
Breaking-Cas Spanish National Center for Biotechnology Yes (over 1000 genomes) Yes Yes (by weights) 4 No User customizable Yes Yes Webserver [3]
Cas-OFFinder Seoul National University Yes Yes No 0-10 No NGG, NRG, NNAGAAW, NNNNGMTT No Yes Webserver Source code [4]
CASTING Caagle Yes Yes No 3 No NGG and NAG No Yes Webserver [5]
CCTop University of Heidelberg Yes Yes Partial 5 (0-5) Yes NGG, NRG, NNGRRT, NNNNGATT, NNAGAAW, NAAAAC Yes Yes Webserver [6]
CHOPCHOP Harvard University Yes Yes Partial 0, 2 No NGG, NNAGAA, NNNNGANN No Yes Webserver [7]
CHOPCHOP v2 University of Bergen Yes Yes Yes 3 (0-3) Yes User customizable Yes Yes Webserver Source code [8]
COD Dayong Guo No No No 0, 3, 5, 8 No NGG and NAG No Yes Webserver -
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 Webserver Source code [9]
CRISPR Design Zhang Lab, MIT Yes No No 4 No NGG and NAG mRNA exons Yes Webserver [10]
CRISPRdirect Database Center for Life Science (DBCLS) Yes (over 200 species) Yes No Any number No NNN Yes Yes Webserver [11]
CRISPR gRNA Design Tool DNA2.0 Yes Yes No 0-10 No NGG, NAG Genbank annotations: Gene, misc_RNA, ncRNA, CDS, exon Yes Webserver -
CRISPR LifePipe Life and Soft Yes Yes Yes 0-5 yes NGG, NGA, NGCG, TTTN, NNGRRT Yes Yes Webserver -
CRISPRscan Giraldez Lab, Yale Yes Yes No 4 Yes NGG, TTTV, TTTN Yes Yes Webserver [12]
CRISPRseek Bioconductor Yes Yes No Any number No User customizable mRNA exons Yes Source code [13]
DESKGEN Desktop Genetics Yes Yes Yes Any number Yes Fully user customizable Yes Yes Webserver [14]
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 Webserver [15]
Genedata Selector Genedata Yes Yes Yes Customizable No Customizable Yes/Pre-loaded Customizable Webserver -
Geneious CRISPR Site Finder Geneious Yes Yes Yes Any number Yes User customizable Yes Yes Geneious [1]
GT-Scan CSIRO & EMBL-ABR Yes Yes Yes 3 (0-3) No User customizable Links to Ensembl genome browser Yes Webserver [16]
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 Webserver Source code Remote batch submission [17]
sgRNA Designer Broad Institute No No No 0 Yes NGG CDS (if searching by transcript ID) Yes Webserver Source code [1]
Synthego Design Tool Synthego Yes (over 120,000 genomes No (Optimized for Knockout) Yes 3 Yes NGG Yes (RefSeq, Ensembl, Gencode) Yes Webserver [18]
TUSCAN CSIRO No No No 0 Yes NGG No Yes Webserver Source Code [19]
VARSCOT CSIRO Yes Yes No 0-8 Yes User customizable No Yes Source Code [20]


  1. ^ a b c d Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, et al. (December 2014). "Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation". Nature Biotechnology. 32 (12): 1262–7. doi:10.1038/nbt.3026. PMC 4262738. PMID 25184501.
  2. ^ a b Chari R, Mali P, Moosburner M, Church GM (September 2015). "Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach". Nature Methods. 12 (9): 823–6. doi:10.1038/nmeth.3473. PMC 5292764. PMID 26167643.
  3. ^ Oliveros JC, Franch M, Tabas-Madrid D, San-León D, Montoliu L, Cubas P, Pazos F (July 2016). "Breaking-Cas-interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes". Nucleic Acids Research. 44 (W1): W267–71. doi:10.1093/nar/gkw407. PMC 4987939. PMID 27166368.
  4. ^ Bae S, Park J, Kim JS (May 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.
  5. ^ Enkler L, Richer D, Marchand AL, Ferrandon D, Jossinet F (October 2016). "Genome engineering in the yeast pathogen Candida glabrata using the CRISPR-Cas9 system". Scientific Reports. 6: 35766. Bibcode:2016NatSR...635766E. doi:10.1038/srep35766. PMC 5073330. PMID 27767081.
  6. ^ 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.
  7. ^ 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.
  8. ^ 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.
  9. ^ 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.
  10. ^ 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.
  11. ^ 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.
  12. ^ 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.
  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. Bibcode:2014PLoSO...9j8424Z. 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. ^ 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.
  16. ^ 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.
  17. ^ 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.
  18. ^ TechCrunch. "Synthego’s genetic toolkit aims to make CRISPR more accessible | May 2017" Retrieved 23 January 2018.
  19. ^ 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.
  20. ^ 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. PMID 31248401.