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.
| 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 | 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] |
References
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- "Desktop Genetics Announces the Launch of DeskGen Gene Editing Platform". American Laboratory. 2015.
- 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.
- 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.
- 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.
- TechCrunch. "Synthego’s genetic toolkit aims to make CRISPR more accessible | May 2017" Retrieved 23 January 2018.
- 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.
- 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.
- 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.
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