Mammalian Genome

, Volume 28, Issue 7–8, pp 283–290 | Cite as

CRISPRtools: a flexible computational platform for performing CRISPR/Cas9 experiments in the mouse

  • Kevin A. Peterson
  • Glen L. Beane
  • Leslie O. Goodwin
  • Peter M. Kutny
  • Laura G. Reinholdt
  • Stephen A. Murray
Article

Abstract

Genome editing using the CRISPR/Cas9 RNA-guided endonuclease system has rapidly become a driving force for discovery in modern biomedical research. This simple yet elegant system has been widely used to generate both loss-of-function alleles and precision knock-in mutations using single-stranded donor oligonucleotides. Our CRISPRtools platform supports both of these applications in order to facilitate the use of CRISPR/Cas9. While there are several tools that facilitate CRISPR/Cas9 design and screen for potential off-target sites, the process is typically performed sequentially on single genes, limiting scalability for large-scale programs. Here, the design principle underlying gene ablation is based upon using paired guides flanking a critical region/exon of interest to create deletions. Guide pairs are rank ordered based upon published efficiency scores and off-target analyses, and reported in a concise format for downstream implementation. The exon deletion strategy simplifies characterization of founder animals and is the strategy employed for the majority of knockouts in the mouse. In proof-of-principle experiments, the effectiveness of this approach is demonstrated using microinjection and electroporation to introduce CRISPR/Cas9 components into mouse zygotes to delete critical exons.

Notes

Acknowledgements

We would like to thank Charles Vejnar, Miguel Moreo-Mateos, and A. Giraldez at Yale University for kindly providing the CRISPRscan code; Susan Kales and Rachel Urban for technical support; Haoyi Wang and Wen-bo Wang for providing insight into electroporation experiments; and the Genetic Engineering Technologies and Transgenic Genotyping Services at the Jackson Laboratory. This work was supported by the Office Of The Director, National Institutes Of Health under Award Number OD011185 and UM1OD023222 (to S.A.M.), and OD010972 (to L.G.R.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Supplementary material

335_2017_9681_MOESM1_ESM.pdf (5.8 mb)
Supplementary material 1 (PDF 5964 KB)
335_2017_9681_MOESM2_ESM.docx (74 kb)
Supplementary material 2 (DOCX 73 KB)
335_2017_9681_MOESM3_ESM.xlsx (36 kb)
Supplementary material 3 (XLSX 36 KB)

References

  1. Bae S, Park J, Kim JS (2014) Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30:1473–1475. doi: 10.1093/bioinformatics/btu048 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bassett AR, Tibbit C, Ponting CP, Liu J-L (2013) Highly efficient targeted mutagenesis of drosophila with the CRISPR/Cas9 system cell Reports 4. doi: 10.1016/j.celrep.2013.06.020
  3. Bradley A et al (2012) The mammalian gene function resource: the International Knockout Mouse Consortium. Mamm Genome 23:580–586. doi: 10.1007/s00335-012-9422-2 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Chen B et al (2013) Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155:1479–1491. doi: 10.1016/j.cell.2013.12.001 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Cheng AW et al (2013) Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res 23:1163–1171. doi: 10.1038/cr.2013.122 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Chu V et al (2016) Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes. BMC Biotechnol 16:4. doi: 10.1186/s12896-016-0234-4 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Cong L et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. doi: 10.1126/science.1231143 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Cradick TJ, Qiu P, Lee CM, Fine EJ, Bao G (2014) COSMID: a web-based tool for identifying and validating CRISPR/Cas off-target sites Mol Ther Nucleic Acids 3:e214. doi: 10.1038/mtna.2014.64 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Dickinson ME, Flenniken AM, Ji X, Teboul L, Wong MD, White JK, Meehan TF, Weninger WJ, Westerberg H, Adissu H, Baker CN, Bower L, Brown JM, Caddle LB, Chiani F, Clary D, Cleak J, Daly MJ, Denegre JM, Doe B, Dolan ME, Edie SM, Fuchs H, Gailus-Durner V, Galli A, Gambadoro A, Gallegos J, Guo S, Horner NR, Hsu C-W, Johnson SJ, Kalaga S, Keith LC, Lanoue L, Lawson TN, Lek M, Mark M, Marschall S, Mason J, McElwee ML, Newbigging S, Nutter LMJ, Peterson KA, Ramirez-Solis R, Rowland DJ, Ryder E, Samocha KE, Seavitt JR, Selloum M, Szoke-Kovacs Z, Tamura M, Trainor AG, Tudose I, Wakana S, Warren J, Wendling O, West DB, Wong L, Yoshiki A, McKay M, Urban B, Lund C, Froeter E, LaCasse T, Mehalow A, Gordon E, Donahue LR, Taft R, Kutney P, Dion S, Goodwin L, Kales S, Urban R, Palmer K, Pertuy F, Bitz D, Weber B, Goetz-Reiner P, Jacobs H, Le Marchand E, El Amri A, El Fertak L, Ennah H, Ali-Hadji D, Ayadi A, Wattenhofer-Donze M, Jacquot S, André P, Birling M-C, Pavlovic G, Sorg T, Morse I, Benso F, Stewart ME, Copley C, Harrison J, Joynson S, Guo R, Qu D, Spring S, Yu L, Ellegood J, Morikawa L, Shang X, Feugas P, Creighton A, Castellanos Penton P, Danisment O, Griggs N, Tudor CL, Green AL, Mazzeo CI, Siragher E, Lillistone C, Tuck E, Gleeson D, Sethi D, Bayzetinova T, Burvill J, Habib B, Weavers L, Maswood R, Miklejewska E, Woods M, Grau E, Newman S, Sinclair C, Brown E, Ayabe S, Iwama M, Murakami A, MacArthur DG, Tocchini-Valentini GP, Gao X, Flicek P, Bradley A, Skarnes WC, Justice MJ, Parkinson HE, Moore M, Wells S, Braun RE, Svenson KL, de Angelis MH, Herault Y, Mohun T, Mallon A-M, Henkelman RM, Brown SDM, Adams DJ, Lloyd KCK, McKerlie C, Beaudet AL, Bućan M, Murray SA (2016) High-throughput discovery of novel developmental phenotypes. Nature 537:(7621):508–514CrossRefPubMedPubMedCentralGoogle Scholar
  10. Doench JG et al (2014) Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol 32:1262–1267. doi: 10.1038/nbt.3026 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Doench JG et al (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34:184–191. doi: 10.1038/nbt.3437 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Gilbert LA et al (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154:442–451. doi: 10.1016/j.cell.2013.06.044 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Heigwer F, Kerr G, Boutros M (2014) E-CRISP: fast CRISPR target site identification. Nat Methods 11:122–123. doi: 10.1038/nmeth.2812 CrossRefPubMedGoogle Scholar
  14. Hodgkins A, Farne A, Perera S, Grego T, Parry-Smith DJ, Skarnes WC, Iyer V (2015) WGE: a CRISPR database for genome engineering. Bioinformatics 31:3078–3080. doi: 10.1093/bioinformatics/btv308 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Hsu PD et al (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832. doi: 10.1038/nbt.2647 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821. doi: 10.1126/science.1225829 CrossRefPubMedGoogle Scholar
  17. Labun K, Montague TG, Gagnon JA, Thyme SB, Valen E (2016) CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering Nucleic Acids Res 44:6 doi: 10.1093/nar/gkw398 CrossRefGoogle Scholar
  18. Li Y et al (2014) CRISPR reveals a distal super-enhancer required for Sox2 expression in mouse embryonic stem cells. PLoS ONE 9:e114485. doi: 10.1371/journal.pone.0114485 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Mali P et al (2013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31:833–838. doi: 10.1038/nbt.2675 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Maquat LE, Li X (2001) Mammalian heat shock p70 and histone H4 transcripts, which derive from naturally intronless genes, are immune to nonsense-mediated decay. RNA 7:445–456CrossRefPubMedPubMedCentralGoogle Scholar
  21. Moreno-Mateos MA, Vejnar CE, Beaudoin J-DD, Fernandez JP, Mis EK, Khokha MK, Giraldez AJ (2015) CRISPR scan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo Nat Methods 12:982–988. doi: 10.1038/nmeth.3543 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Oliveros JC, Franch M, Tabas-Madrid D, San-Leon D, Montoliu L, Cubas P, Pazos F (2016) Breaking-Cas-interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes. Nucleic Acids Res 44:W267–W271. doi: 10.1093/nar/gkw407 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–1183. doi: 10.1016/j.cell.2013.02.022 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Qin W et al (2015) Efficient CRISPR/Cas9-mediated genome editing in mice by zygote electroporation of nuclease. Genetics 200:423–430. doi: 10.1534/genetics.115.176594 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Richardson CD, Ray GJ, DeWitt MA, Curie GL, Corn JE (2016) Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol 34:339–344. doi: 10.1038/nbt.3481 CrossRefPubMedGoogle Scholar
  26. Skarnes WC et al (2011) A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474:337–342. doi: 10.1038/nature10163 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918. doi: 10.1016/j.cell.2013.04.025 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Wang W et al (2016) Delivery of Cas9 protein into mouse zygotes through a series of electroporation dramatically increases the efficiency of model creation. J Genet Genom 43:319–327. doi: 10.1016/j.jgg.2016.02.004 CrossRefGoogle Scholar
  29. Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154:1370–1379. doi: 10.1016/j.cell.2013.08.022 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Zetsche B et al (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:759–771. doi: 10.1016/j.cell.2015.09.038 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Zhang X, Choi PS, Francis JM, Imielinski M, Watanabe H, Cherniack AD, Meyerson M (2016) Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers. Nat Genet 48:176–182. doi: 10.1038/ng.3470 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.The Jackson LaboratoryBar HarborUSA

Personalised recommendations