Skip to main content
SpringerLink
Log in

Rapid and Simple Screening of CRISPR Guide RNAs (gRNAs) in Cultured Cells Using Adeno-Associated Viral (AAV) Vectors

  • Protocol
  • First Online:
CRISPR Gene Editing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1961))

Abstract

Genome editing reagents including the recently introduced CRISPR/Cas9 system have become established and widely used molecular tools to answer fundamental biological questions and to target and treat genetic diseases. The CRISPR system, originally derived from bacteria and archaea, can be delivered into cells using different techniques, comprising (1) transfection of mRNA or plasmid DNA, (2) electroporation of plasmid DNA or the Cas9 protein in a complex with a g(uide)RNA, or (3) use of nonviral or viral vectors. Among the latter, Adeno-associated viruses (AAVs) are particularly attractive owing to many favorable traits: (1) their apathogenicity and episomal persistence, (2) the ease of virus production and purification, (3) the safe handling under lowest biosafety level 1 conditions, and (4) the availability of numerous natural serotypes and synthetic capsid variants with distinct cell specificities. Here, we describe a fast and simple protocol for small-scale packaging of CRISPR/Cas9 components into AAV vectors. To showcase its potential, we employ this method for screening of gRNAs targeting the murine miR-122 locus in Hepa1–6 cells (using AAV serotype 6, AAV6) or the 5′LTR of the human immunodeficiency virus (HIV) in HeLaP4-NLtr cells (using a synthetic AAV9 variant). We furthermore provide a detailed protocol for large-scale production of purified AAV/CRISPR vector stocks that permit higher cleavage efficiencies in vitro and are suitable for direct in vivo applications.

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 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 199.99
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. Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. https://doi.org/10.1126/science.1231143

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826. https://doi.org/10.1126/science.1232033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Barrangou R, Doudna JA (2016) Applications of CRISPR technologies in research and beyond. Nat Biotechnol 34:933–941. https://doi.org/10.1038/nbt.3659

    Article  CAS  PubMed  Google Scholar 

  4. 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. https://doi.org/10.1126/science.1225829

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Boettcher M, McManus MT (2015) Choosing the right tool for the job: RNAi, TALEN, or CRISPR. Mol Cell 58:575–585. https://doi.org/10.1016/j.molcel.2015.04.028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346:1258096. https://doi.org/10.1126/science.1258096

    Article  CAS  PubMed  Google Scholar 

  7. Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278. https://doi.org/10.1016/j.cell.2014.05.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Porteus M (2016) Genome editing: a new approach to human therapeutics. Annu Rev Pharmacol Toxicol 56:163–190. https://doi.org/10.1146/annurev-pharmtox-010814-124454

    Article  CAS  PubMed  Google Scholar 

  9. Dominguez AA, Lim WA, Qi LS (2016) Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat Rev Mol Cell Biol 17:5–15. https://doi.org/10.1038/nrm.2015.2

    Article  CAS  PubMed  Google Scholar 

  10. Gilbert LA, Horlbeck MA, Adamson B et al (2014) Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159:647–661. https://doi.org/10.1016/j.cell.2014.09.029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 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. https://doi.org/10.1016/j.cell.2013.02.022

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, Vale RD (2014) A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159:635–646. https://doi.org/10.1016/j.cell.2014.09.039

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kearns NA, Pham H, Tabak B, Genga RM, Silverstein NJ, Garber M, Maehr R (2015) Functional annotation of native enhancers with a Cas9-histone demethylase fusion. Nat Methods 12:401–403. https://doi.org/10.1038/nmeth.3325

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Thakore PI, D’Ippolito AM, Song L et al (2015) Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat Methods 12:1143–1149. https://doi.org/10.1038/nmeth.3630

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Grimm D, Zolotukhin S (2015) E Pluribus Unum: 50 years of research, millions of viruses, and one goal--tailored acceleration of AAV evolution. Mol Ther 23:1819–1831. https://doi.org/10.1038/mt.2015.173

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Schmidt F, Grimm D (2015) CRISPR genome engineering and viral gene delivery: a case of mutual attraction. Biotechnol J 10:258–272. https://doi.org/10.1002/biot.201400529

    Article  CAS  PubMed  Google Scholar 

  17. Epstein BE, Schaffer DV (2017) Combining engineered nucleases with adeno-associated viral vectors for therapeutic gene editing. Adv Exp Med Biol 1016:29–42. https://doi.org/10.1007/978-3-319-63904-8_2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Schmelas C, Grimm D (2018) Split Cas9, Not hairs - advancing the therapeutic index of CRISPR Technology. Biotechnol J 13(9):e1700432. https://doi.org/10.1002/biot.201700432

    Article  CAS  PubMed  Google Scholar 

  19. Senis E, Fatouros C, Grosse S et al (2014) CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox. Biotechnol J 9:1402–1412. https://doi.org/10.1002/biot.201400046

    Article  CAS  PubMed  Google Scholar 

  20. Swiech L, Heidenreich M, Banerjee A et al (2015) In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol 33:102–106. https://doi.org/10.1038/nbt.3055

    Article  CAS  PubMed  Google Scholar 

  21. Chew WL, Tabebordbar M, Cheng JK et al (2016) A multifunctional AAV-CRISPR-Cas9 and its host response. Nat Methods 13:868–874. https://doi.org/10.1038/nmeth.3993

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Long C, Amoasii L, Mireault AA et al (2016) Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 351:400–403. https://doi.org/10.1126/science.aad5725

    Article  CAS  PubMed  Google Scholar 

  23. Nelson CE, Hakim CH, Ousterout DG et al (2016) In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351:403–407. https://doi.org/10.1126/science.aad5143

    Article  CAS  PubMed  Google Scholar 

  24. Tabebordbar M, Zhu K, Cheng JKW et al (2016) In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 351:407–411. https://doi.org/10.1126/science.aad5177

    Article  CAS  PubMed  Google Scholar 

  25. Chow RD, Guzman CD, Wang G et al (2017) AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma. Nat Neurosci 20:1329–1341. https://doi.org/10.1038/nn.4620

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Wang G, Chow RD, Ye L et al (2018) Mapping a functional cancer genome atlas of tumor suppressors in mouse liver using AAV-CRISPR-mediated direct in vivo screening. Sci Adv 4:eaao5508. https://doi.org/10.1126/sciadv.aao5508

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Platt RJ, Chen S, Zhou Y et al (2014) CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159:440–455. https://doi.org/10.1016/j.cell.2014.09.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ran FA, Cong L, Yan WX et al (2015) In vivo genome editing using Staphylococcus aureus Cas9. Nature 520:186–191. https://doi.org/10.1038/nature14299

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Grimm D, Streetz KL, Jopling CL et al (2006) Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441:537–541. https://doi.org/10.1038/nature04791

    Article  CAS  PubMed  Google Scholar 

  30. McCarty DM, Fu H, Monahan PE, Toulson CE, Naik P, Samulski RJ (2003) Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo. Gene Ther 10:2112–2118. https://doi.org/10.1038/sj.gt.3302134

    Article  CAS  PubMed  Google Scholar 

  31. McCarty DM, Monahan PE, Samulski RJ (2001) Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Ther 8:1248–1254. https://doi.org/10.1038/sj.gt.3301514

    Article  CAS  PubMed  Google Scholar 

  32. Zolotukhin S, Byrne BJ, Mason E et al (1999) Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther 6:973–985. https://doi.org/10.1038/sj.gt.3300938

    Article  CAS  PubMed  Google Scholar 

  33. Kotterman MA, Schaffer DV (2014) Engineering adeno-associated viruses for clinical gene therapy. Nat Rev Genet 15:445–451. https://doi.org/10.1038/nrg3742

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zincarelli C, Soltys S, Rengo G, Rabinowitz JE (2008) Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther 16:1073–1080. https://doi.org/10.1038/mt.2008.76

    Article  CAS  PubMed  Google Scholar 

  35. Weinmann J, Grimm D (2017) Next-generation AAV vectors for clinical use: an ever-accelerating race. Virus Genes 53(5):707–713. https://doi.org/10.1007/s11262-017-1502-7

    Article  CAS  PubMed  Google Scholar 

  36. Ellis BL, Hirsch ML, Barker JC, Connelly JP, Steininger RJ 3rd, Porteus MH (2013) A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype. Virol J 10:74. https://doi.org/10.1186/1743-422X-10-74

    Article  PubMed  PubMed Central  Google Scholar 

  37. Lock M, Alvira M, Vandenberghe LH, Samanta A, Toelen J, Debyser Z, Wilson JM (2010) Rapid, simple, and versatile manufacturing of recombinant adeno-associated viral vectors at scale. Hum Gene Ther 21:1259–1271. https://doi.org/10.1089/hum.2010.055

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Strobel B, Miller FD, Rist W, Lamla T (2015) Comparative analysis of cesium chloride- and iodixanol-based purification of recombinant adeno-associated viral vectors for preclinical applications. Hum Gene Ther Methods 26:147–157. https://doi.org/10.1089/hgtb.2015.051

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Guschin DY, Waite AJ, Katibah GE, Miller JC, Holmes MC, Rebar EJ (2010) A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol 649:247–256. https://doi.org/10.1007/978-1-60761-753-2_15

    Article  CAS  PubMed  Google Scholar 

  40. Ran FA, Hsu PD, Lin CY et al (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154:1380–1389. https://doi.org/10.1016/j.cell.2013.08.021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. BeckmanCoulter (2014) Using K-factor to compare rotor efficiency. Technical guide CENT-66APP0. www.beckmancoulter.com

    Google Scholar 

Download references

Acknowledgments

J.F. and D.G. are grateful for funding from the Cystic Fibrosis Foundation Therapeutics (CFFT, grant number GRIMM15XX0) as well as from the Heidelberg Biosciences International Graduate School HBIGS at Heidelberg University. D.G. acknowledges funding by the Collaborative Research Center SFB1129 (German Research Foundation [DFG], project TP2), the Cluster of Excellence CellNetworks (DFG; EXC81), and the Transregional DFG Collaborative Research Center TRR179 (project TP18, Projektnummer 272983813). M.N. and D.G. are grateful for additional funding from the German Center for Infection Research (DZIF; TTU-HIV 04.803). Moreover, M.N. appreciates funding from the Heidelberg Innovation fund FRONTIER (project “A CRISPR way to eradicate viral pathogens”).

Author information

Author notes

  1. Julia Fakhiri and Manuela Nickl contributed equally to this work.

Authors and Affiliations

  1. Department of Infectious Diseases/Virology, Heidelberg University Hospital, Cluster of Excellence Cell Networks, Heidelberg, Germany

    Julia Fakhiri, Manuela Nickl & Dirk Grimm

  2. BioQuant Center, University of Heidelberg, Heidelberg, Germany

    Julia Fakhiri, Manuela Nickl & Dirk Grimm

  3. German Center for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany

    Manuela Nickl & Dirk Grimm

Authors
  1. Julia Fakhiri
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Manuela Nickl
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dirk Grimm
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dirk Grimm .

Editor information

Editors and Affiliations

  1. Department of Biomedicine, Aarhus University, Aarhus, Denmark

    Yonglun Luo

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Fakhiri, J., Nickl, M., Grimm, D. (2019). Rapid and Simple Screening of CRISPR Guide RNAs (gRNAs) in Cultured Cells Using Adeno-Associated Viral (AAV) Vectors. In: Luo, Y. (eds) CRISPR Gene Editing. Methods in Molecular Biology, vol 1961. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9170-9_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9170-9_8

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9169-3

  • Online ISBN: 978-1-4939-9170-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation