CRISPR/Cas tools

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.

CRISPR-Cas

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.

Table

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]
WGE	Wellcome Sanger Institute	Yes, human and mouse reference genomes	Yes	Yes	4 (0-4)	No	NGG	Yes	Yes	[26]

References

1. 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. 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. Xiang X, Corsi, GI, Anthon C, Qu K, Pan X, Liang X, Han P, Dong Z, Liu L, Zhong J, Ma T, Wang J, Zhang X, Jiang H, Xu F, Liu X, Xu X, Wang J, Yang H, Bolund L, Church GM, Lin L, Gorodkin J, Luo Y (May 2021). "Enhancing CRISPR-Cas9 gRNA efficiency prediction by data integration and deep learning". Nature Communications. 12 (1): 3238. Bibcode:2021NatCo..12.3238X. doi:10.1038/s41467-021-23576-0. PMC 8163799. PMID 34050182.
4. Alkan F, Wenzel A, Anthon C, Havgaard JH, Gorodkin J (October 2018). "CRISPR-Cas9 off-targeting assessment with nucleic acid duplex energy parameters". Genome Biology. 19 (177): 177. doi:10.1186/s13059-018-1534-x. PMC 6203265. PMID 30367669.
5. Liang X, Potter J, Kumar S, Ravinder N, Chesnut JD (January 2017). "Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA". Journal of Biotechnology. 241: 136–146. doi:10.1016/j.jbiotec.2016.11.011. PMID 27845164.
6. 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.
7. 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.
8. 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.
9. Pedersen LE, Ronda C, Hansen HG, Kallehauge TB, Betenbaugh MJ, Nielsen AT, Kildegaard HF (August 2014). "Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy". Biotechnology and Bioengineering. 111 (8): 1604–1616. doi:10.1002/bit.25233. PMC 4312910. PMID 24827782.
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.
22. TechCrunch. "Synthego’s genetic toolkit aims to make CRISPR more accessible | May 2017" Retrieved 23 January 2018.
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. PMC 8975720. PMID 35365834. S2CID 235700000.
26. Hodgkins A, Farne A, Perera S, Grego T, Parry-Smith DJ, Skarnes WC, Iyer V (September 2015). "WGE: a CRISPR database for genome engineering". Bioinformatics. 31 (18): 3078–3080. doi:10.1093/bioinformatics/btv308. PMID 25979474.