CRISPR/Cas tools explained

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.__TOC__

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, 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 NameProviderSearches whole genome for targetsReturns all targets of genomeSeed span and location can be definedMaximum number of mismatches supportedPredicts gRNA activityAvailable Protospacer adjacent motif (PAM) sequencesAnnotation is reportedgRNA suggestion or scoringReferences
CRISPRon, CRISPRoffCenter for non-coding RNA in Technology and Health, University of CopenhagenYesYesYesAllYesNGG, NGA, NAGYesYes,[3] [4]
Invitrogen TrueDesign Genome EditorThermo Fisher ScientificYesYesNo3NoNGGYesYes[5]
Breaking-CasSpanish National Center for BiotechnologyYes (over 1000 genomes)YesYes (by weights)4NoUser customizableYesYes[6]
Cas-OFFinder Seoul National UniversityYes Yes No 0-10NoNGG, NRG, NNAGAAW, NNNNGMTTNo Yes [7]
CASTINGCaagleYesYesNo3NoNGG and NAGNoYes[8]
CRISPy Technical University of DenmarkYes Yes No All No NGG Yes Yes [9]
CCTop University of HeidelbergYes Yes Partial 5 (0-5)YesNGG, NRG, NNGRRT, NNNNGATT, NNAGAAW, NAAAAC Yes Yes [10]
CHOPCHOP Harvard UniversityYes Yes Partial 0, 2NoNGG, NNAGAA, NNNNGANN No Yes [11]
CHOPCHOP v2 University of BergenYes Yes Yes 3 (0-3)YesUser customizable Yes Yes [12]
CRISPOR University of California, Santa Cruz TEFORYes (over 200 genomes) Yes No 4YesNGG, NGA, NGCG, NNAGAA, NGGNG, NNGRRT, NNNRRT, NNNNGMTT, NNNNACA, TTTN Yes Yes [13]
CRISPR Design Zhang Lab, MITYes No No 4NoNGG and NAG mRNA exons Yes [14]
CRISPRdirect Database Center for Life Science (DBCLS) Yes (over 200 species) Yes No Any numberNoNNN Yes Yes [15]
CRISPRscan Giraldez Lab, YaleYes Yes No 4YesNGG, TTTV, TTTN Yes Yes [16]
CRISPRseek BioconductorYes Yes No Any numberNoUser customizable mRNA exons Yes [17]
DESKGEN Desktop GeneticsYes Yes Yes Any numberYesFully user customizable Yes Yes [18]
GuideScan GuideScanYes Yes Yes 3 on website and customizable with command lineYesNGG/NAG on website and customizable with command line Yes Yes [19]
GT-ScanCSIRO & YesYesYes3 (0-3)NoUser customizableLinks to Ensembl genome browserYes[20]
Off-Spotter Thomas Jefferson UniversityYes Yes Yes 0-5NGG, 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 InstituteNo No No 0YesNGG CDS (if searching by transcript ID) Yes
Synthego Design ToolSynthegoYes (over 120,000 genomes)No (Optimized for Knockout)Yes3YesNGGYes (RefSeq, Ensembl, Gencode)Yes[22]
TUSCAN CSIRONo No No 0 Yes NGG No Yes [23]
VARSCOT CSIROYes Yes No 0-8 Yes User customizable No Yes [24]
CRISPR Targeted Gene DesignerHorizon DiscoveryYes, Multipleyesyes4YesNGG, NNGRRT, YTTV, otherYesYes(21)
GuideMakerUnited States Department of Agriculture, Agricultural Research ServiceYes, any user supplied genomeYesYes0-5YesAny PAM site and PAM orientationYesYes[25]

Notes and References

  1. 6. Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, Sullender M, Ebert BL, Xavier RJ, Root DE. December 2014. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nature Biotechnology. 32. 12. 1262–7. 10.1038/nbt.3026. 4262738. 25184501.
  2. Chari R, Mali P, Moosburner M, Church GM . Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach . Nature Methods . 12 . 9 . 823–6 . September 2015 . 26167643 . 5292764 . 10.1038/nmeth.3473 .
  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 . Enhancing CRISPR-Cas9 gRNA efficiency prediction by data integration and deep learning . Nature Communications . 12 . 1 . May 2021 . 3238 . 10.1038/s41467-021-23576-0 . 34050182 . 8163799 . 2021NatCo..12.3238X . free .
  4. Alkan F, Wenzel A, Anthon C, Havgaard JH, Gorodkin J . CRISPR-Cas9 off-targeting assessment with nucleic acid duplex energy parameters . Genome Biology . 19 . 177 . October 2018 . 177 . 30367669 . 6203265 . 10.1186/s13059-018-1534-x . free.
  5. Liang X, Potter J, Kumar S, Ravinder N, Chesnut JD . 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 . January 2017 . 27845164 . 10.1016/j.jbiotec.2016.11.011 . free .
  6. Oliveros JC, Franch M, Tabas-Madrid D, San-León D, Montoliu L, Cubas P, Pazos F . Breaking-Cas-interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes . Nucleic Acids Research . 44 . W1 . W267-71 . July 2016 . 27166368 . 4987939 . 10.1093/nar/gkw407 .
  7. Bae S, Park J, Kim JS . Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases . Bioinformatics . 30 . 10 . 1473–5 . May 2014 . 24463181 . 4016707 . 10.1093/bioinformatics/btu048 .
  8. Enkler L, Richer D, Marchand AL, Ferrandon D, Jossinet F . Genome engineering in the yeast pathogen Candida glabrata using the CRISPR-Cas9 system . Scientific Reports . 6 . 35766 . October 2016 . 27767081 . 5073330 . 10.1038/srep35766 . 2016NatSR...635766E .
  9. Pedersen LE, Ronda C, Hansen HG, Kallehauge TB, Betenbaugh MJ, Nielsen AT, Kildegaard HF . Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy . Biotechnology and Bioengineering . 111 . 8 . 1604–1616 . August 2014 . 24827782 . 4312910 . 10.1002/bit.25233 .
  10. Stemmer M, Thumberger T, Del Sol Keyer M, Wittbrodt J, Mateo JL . CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool . PLOS ONE . 10 . 4 . e0124633 . 2015 . 25909470 . 4409221 . 10.1371/journal.pone.0124633 . 2015PLoSO..1024633S . free .
  11. Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E . CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing . Nucleic Acids Research . 42 . Web Server issue . W401-7 . July 2014 . 24861617 . 4086086 . 10.1093/nar/gku410 .
  12. Labun K, Montague TG, Gagnon JA, Thyme SB, Valen E . CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering . Nucleic Acids Research . 44 . W1 . W272–6 . July 2016 . 27185894 . 4987937 . 10.1093/nar/gkw398 .
  13. Haeussler M, Schönig K, Eckert H, Eschstruth A, Mianné J, Renaud JB, Schneider-Maunoury S, Shkumatava A, Teboul L, Kent J, Joly JS, Concordet JP . 6 . Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR . Genome Biology . 17 . 1 . 148 . July 2016 . 27380939 . 4934014 . 10.1186/s13059-016-1012-2 . free .
  14. 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 . 6 . DNA targeting specificity of RNA-guided Cas9 nucleases . Nature Biotechnology . 31 . 9 . 827–32 . September 2013 . 23873081 . 3969858 . 10.1038/nbt.2647 . 1721.1/102691 .
  15. Naito Y, Hino K, Bono H, Ui-Tei K . CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites . Bioinformatics . 31 . 7 . 1120–3 . April 2015 . 25414360 . 4382898 . 10.1093/bioinformatics/btu743 .
  16. Moreno-Mateos MA, Vejnar CE, Beaudoin JD, Fernandez JP, Mis EK, Khokha MK, Giraldez AJ . CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo . Nature Methods . 12 . 10 . 982–8 . October 2015 . 26322839 . 4589495 . 10.1038/nmeth.3543 .
  17. Zhu LJ, Holmes BR, Aronin N, Brodsky MH . CRISPRseek: a bioconductor package to identify target-specific guide RNAs for CRISPR-Cas9 genome-editing systems . PLOS ONE . 9 . 9 . e108424 . 2014 . 25247697 . 4172692 . 10.1371/journal.pone.0108424 . 2014PLoSO...9j8424Z . free .
  18. News: 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 . GuideScan software for improved single and paired CRISPR guide RNA design . Nature Biotechnology . 35 . 4 . 347–349 . April 2017 . 28263296 . 5607865 . 10.1038/nbt.3804 .
  20. O'Brien A, Bailey TL . GT-Scan: identifying unique genomic targets . Bioinformatics . 30 . 18 . 2673–5 . September 2014 . 24860161 . 4155256 . 10.1093/bioinformatics/btu354 .
  21. Pliatsika V, Rigoutsos I . "Off-Spotter": very fast and exhaustive enumeration of genomic lookalikes for designing CRISPR/Cas guide RNAs . Biology Direct . 10 . 1 . 4 . January 2015 . 25630343 . 4326336 . 10.1186/s13062-015-0035-z . free .
  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 . High Activity Target-Site Identification Using Phenotypic Independent CRISPR-Cas9 Core Functionality . The CRISPR Journal . 1 . 2 . 182–190 . April 2018 . 31021206 . 10.1089/crispr.2017.0021 .
  24. Wilson LO, Hetzel S, Pockrandt C, Reinert K, Bauer DC . VARSCOT: variant-aware detection and scoring enables sensitive and personalized off-target detection for CRISPR-Cas9 . BMC Biotechnology . 19 . 1 . 40 . June 2019 . 31248401 . 6598273 . 10.1186/s12896-019-0535-5 . free .
  25. Poudel R, Rodriguez LT, Reisch CR, Rivers AR . GuideMaker: Software to design CRISPR-Cas guide RNA pools in non-model genomes . GigaScience . 11 . April 2022 . 35365834 . 10.1093/gigascience/giac007 . 8975720 . 235700000 . free .

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