Skip to main content
SpringerLink
Log in

Cloud-Based Design of Short Guide RNA (sgRNA) Libraries for CRISPR Experiments

  • Protocol
  • First Online:
CRISPR Guide RNA Design

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2162))

  • 2484 Accesses

Abstract

CRISPR/Cas-based genome editing in any biological application requires the evaluation of suitable genomic target sites to design efficient reagents. Considerations for the design of short guide (sg) RNAs include the assessment of possible off-target activities, the prediction of on-target efficacies and mutational outcome. Manual design of sgRNAs taking into account these parameters, however, remains a difficult task. Thus, computational tools to design sgRNA reagents from small scale to genome-wide libraries have been developed that assist during all steps of the design process. Here, we will describe practical guidance for the sgRNA design process using the web-based tool E-CRISP used in the design of individual sgRNAs. E-CRISP (www.e-crisp.org) has been the first web-based sgRNA design tool and uniquely features simple, yet efficient, scoring schemes in combination with fast evaluation and simple usage. We will also discuss the installation of a dockerized version of CRISPR Library Designer (CLD) that can be deployed locally or in the cloud to support the end-to-end design of sgRNA libraries for more than 50 different organisms. CLD was built upon E-CRISP to further increase the scope of sgRNA design to more experimental modalities (CRISPRa/i, Cas12a, all possible protospacer adjacency motifs) offering the same flexibility as E-CRISP, plus the scalability through local and cloud installation. Together, these tools facilities the design of small and large-scale CRISPR/Cas experiments.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Doench J, Am G (2018) I ready for CRISPR? A user’s guide to genetic screens. Nat Rev Genet 19:67–80

    Article  CAS  Google Scholar 

  2. Zhan T, Rindtorff N, Betge J, Ebert MP, Boutros M (2018) CRISPR/Cas9 for cancer research and therapy. Semin Cancer Biol. https://doi.org/10.1016/j.semcancer.2018.04.001

  3. Cong L et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:823–826

    Article  Google Scholar 

  4. Wang T et al (2017) Gene essentiality profiling reveals gene networks and synthetic lethal interactions with oncogenic Ras. Cell 168:890–903.e15

    Article  CAS  Google Scholar 

  5. Shalem O et al (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343:84–87

    Article  CAS  Google Scholar 

  6. LaFountaine JS, Fathe K, Smyth HDC (2015) Delivery and therapeutic applications of gene editing technologies ZFNs, TALENs, and CRISPR/Cas9. Int J Pharm 494:180–194

    Article  CAS  Google Scholar 

  7. Chuai G-H, Wang Q-L, Liu Q (2017) In silico meets in vivo: towards computational CRISPR-Based sgRNA design. Trends Biotechnol 35:12–21

    Article  CAS  Google Scholar 

  8. Doench JG et al (2014) Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol 32:1262–1267

    Article  CAS  Google Scholar 

  9. Graham DB, Root DE (2015) Resources for the design of CRISPR gene editing experiments. Genome Biol 16:260

    Article  Google Scholar 

  10. Cui Y, Xu J, Cheng M, Liao X, Peng S (2018) Review of CRISPR/Cas9 sgRNA design tools. Interdiscip Sci 10:455–465

    Article  CAS  Google Scholar 

  11. Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E (2014) CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res 42:W401–W407

    Article  CAS  Google Scholar 

  12. 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:e0124633

    Article  Google Scholar 

  13. Haeussler M et al (2016) Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol 17

    Google Scholar 

  14. Perez AR et al (2017) GuideScan software for improved single and paired CRISPR guide RNA design. Nat Biotechnol 35:347–349

    Article  CAS  Google Scholar 

  15. Heigwer F, Kerr G, Boutros M (2014) E-CRISP: fast CRISPR target site identification. Nat Methods 11:122–123

    Article  CAS  Google Scholar 

  16. Heigwer F et al (2016) CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries. Genome Biol 17:55

    Article  Google Scholar 

  17. Doench JG et al (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34:184–191

    Article  CAS  Google Scholar 

  18. Chuai G et al (2018) DeepCRISPR: optimized CRISPR guide RNA design by deep learning. Genome Biol 19:80

    Article  Google Scholar 

  19. Dang Y et al (2015) Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency. Genome Biol 16:280

    Article  Google Scholar 

  20. Singh D, Sternberg SH, Fei J, Doudna JA, Ha T (2016) Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9. Nat Commun 7:12778

    Article  CAS  Google Scholar 

  21. Hilton IB, Gersbach CA (2015) Enabling functional genomics with genome engineering. Genome Res 25:1442–1455

    Article  CAS  Google Scholar 

  22. Horlbeck MA et al (2016) Nucleosomes impede Cas9 access to DNA in vivo and in vitro. elife 5:e12677

    Article  Google Scholar 

  23. Shi J et al (2015) Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains. Nat Biotechnol 33:661–667

    Article  CAS  Google Scholar 

  24. Uusi-Mäkelä MIE et al (2018) Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (Danio rerio). PLoS One 13:e0196238

    Article  Google Scholar 

  25. Bae S, Kweon J, Kim HS, Kim J-S (2014) Microhomology-based choice of Cas9 nuclease target sites. Nat Methods 11:705–706

    Article  CAS  Google Scholar 

  26. Tsai SQ et al (2015) GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33:1–12. https://doi.org/10.1038/nbt.3117

    Article  CAS  Google Scholar 

  27. Yang H et al (2018) Base editing generates substantial off-target single nucleotide variants. bioRxiv 480145. https://doi.org/10.1101/480145

  28. Kuscu C, Arslan S, Singh R, Thorpe J, Adli M (2014) Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat Biotechnol 32:677–683

    Article  CAS  Google Scholar 

  29. Pattanayak V et al (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol 31:839–843

    Article  CAS  Google Scholar 

  30. Kleinstiver BP et al (2016) High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature. https://doi.org/10.1038/nature16526

  31. Wu X, Kriz AJ, Sharp PA (2014) Target specificity of the CRISPR-Cas9 system. Quant Biol 2:59–70

    Article  CAS  Google Scholar 

  32. Bolukbasi MF, Gupta A, Wolfe SA (2016) Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery. Nat Methods 13:41–50

    Article  CAS  Google Scholar 

  33. Weiner A (2018) Cloning guides to lentiCRISPR v2. https://doi.org/10.17504/protocols.io.qx3dxqn

  34. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25

    Article  Google Scholar 

  35. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359

    Article  CAS  Google Scholar 

  36. MacPherson CR, Scherf A (2015) Flexible guide-RNA design for CRISPR applications using Protospacer Workbench. Nat Biotechnol 33:805–806

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Heidelberg, Germany

    Florian Heigwer & Michael Boutros

  2. Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany

    Florian Heigwer & Michael Boutros

Authors
  1. Florian Heigwer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael Boutros
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael Boutros .

Editor information

Editors and Affiliations

  1. Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    Tudor A. Fulga

  2. Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    David J. H. F. Knapp

  3. Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    Quentin R. V. Ferry

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Heigwer, F., Boutros, M. (2021). Cloud-Based Design of Short Guide RNA (sgRNA) Libraries for CRISPR Experiments. In: Fulga, T.A., Knapp, D.J.H.F., Ferry, Q.R.V. (eds) CRISPR Guide RNA Design. Methods in Molecular Biology, vol 2162. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0687-2_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0687-2_1

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0686-5

  • Online ISBN: 978-1-0716-0687-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation