Skip to main content
SpringerLink
Log in

CRISPR-Mediated Mutagenesis of Long Noncoding RNAs

  • Protocol
Functional Analysis of Long Non-Coding RNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2254))

Abstract

Functional characterizations and molecular dissections of long noncoding RNAs (lncRNAs) are critical to understand their involvement in the cellular regulatory network. LncRNAs exert their effects through functional RNA domains that interact with other molecules, including proteins, DNA, and RNA. Here, we describe experimental procedures for generating genomic deletions in a human haploid cell line using the CRISPR/Cas9 system. This method can be applied to examine functions of lncRNAs and their domains by establishing knockout and partial deletion mutant cell lines. In addition, we describe a CRISPR-mediated knockin method for artificial tethering of partner RNA-binding proteins to lncRNAs and its use to validate lncRNA-mediated functions.

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 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Djebali S, Davis CA, Merkel A, Dobin A, Lassmann T, Mortazavi A, Tanzer A, Lagarde J, Lin W, Schlesinger F, Xue C, Marinov GK, Khatun J, Williams BA, Zaleski C, Rozowsky J, Roder M, Kokocinski F, Abdelhamid RF, Alioto T, Antoshechkin I, Baer MT, Bar NS, Batut P, Bell K, Bell I, Chakrabortty S, Chen X, Chrast J, Curado J, Derrien T, Drenkow J, Dumais E, Dumais J, Duttagupta R, Falconnet E, Fastuca M, Fejes-Toth K, Ferreira P, Foissac S, Fullwood MJ, Gao H, Gonzalez D, Gordon A, Gunawardena H, Howald C, Jha S, Johnson R, Kapranov P, King B, Kingswood C, Luo OJ, Park E, Persaud K, Preall JB, Ribeca P, Risk B, Robyr D, Sammeth M, Schaffer L, See LH, Shahab A, Skancke J, Suzuki AM, Takahashi H, Tilgner H, Trout D, Walters N, Wang H, Wrobel J, Yu Y, Ruan X, Hayashizaki Y, Harrow J, Gerstein M, Hubbard T, Reymond A, Antonarakis SE, Hannon G, Giddings MC, Ruan Y, Wold B, Carninci P, Guigo R, Gingeras TR (2012) Landscape of transcription in human cells. Nature 489:101–108

    Article  CAS  Google Scholar 

  2. Iyer MK, Niknafs YS, Malik R, Singhal U, Sahu A, Hosono Y, Barrette TR, Prensner JR, Evans JR, Zhao S, Poliakov A, Cao X, Dhanasekaran SM, Wu YM, Robinson DR, Beer DG, Feng FY, Iyer HK, Chinnaiyan AM (2015) The landscape of long noncoding RNAs in the human transcriptome. Nat Genet 47:199–208

    Article  CAS  Google Scholar 

  3. Hon CC, Ramilowski JA, Harshbarger J, Bertin N, Rackham OJ, Gough J, Denisenko E, Schmeier S, Poulsen TM, Severin J, Lizio M, Kawaji H, Kasukawa T, Itoh M, Burroughs AM, Noma S, Djebali S, Alam T, Medvedeva YA, Testa AC, Lipovich L, Yip CW, Abugessaisa I, Mendez M, Hasegawa A, Tang D, Lassmann T, Heutink P, Babina M, Wells CA, Kojima S, Nakamura Y, Suzuki H, Daub CO, de Hoon MJ, Arner E, Hayashizaki Y, Carninci P, Forrest AR (2017) An atlas of human long non-coding RNAs with accurate 5′ ends. Nature 543:199–204

    Article  CAS  Google Scholar 

  4. Engreitz JM, Ollikainen N, Guttman M (2016) Long non-coding RNAs: spatial amplifiers that control nuclear structure and gene expression. Nat Rev Mol Cell Biol 17:756–770

    Article  CAS  Google Scholar 

  5. Quinn JJ, Chang HY (2016) Unique features of long non-coding RNA biogenesis and function. Nat Rev Genet 17:47–62

    Article  CAS  Google Scholar 

  6. Kopp F, Mendell JT (2018) Functional classification and experimental dissection of long noncoding RNAs. Cell 172:393–407

    Article  CAS  Google Scholar 

  7. Yamazaki T, Souquere S, Chujo T, Kobelke S, Chong YS, Fox AH, Bond CS, Nakagawa S, Pierron G, Hirose T (2018) Functional domains of NEAT1 architectural lncRNA induce paraspeckle assembly through phase separation. Mol Cell 70:1038–1053.e7

    Article  CAS  Google Scholar 

  8. Chu C, Zhang QC, da Rocha ST, Flynn RA, Bharadwaj M, Calabrese JM, Magnuson T, Heard E, Chang HY (2015) Systematic discovery of Xist RNA binding proteins. Cell 161:404–416

    Article  CAS  Google Scholar 

  9. McHugh CA, Chen CK, Chow A, Surka CF, Tran C, McDonel P, Pandya-Jones A, Blanco M, Burghard C, Moradian A, Sweredoski MJ, Shishkin AA, Su J, Lander ES, Hess S, Plath K, Guttman M (2015) The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature 521:232–236

    Article  CAS  Google Scholar 

  10. Engreitz JM, Haines JE, Perez EM, Munson G, Chen J, Kane M, McDonel PE, Guttman M, Lander ES (2016) Local regulation of gene expression by lncRNA promoters, transcription and splicing. Nature 539:452–455

    Article  CAS  Google Scholar 

  11. Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278

    Article  CAS  Google Scholar 

  12. Essletzbichler P, Konopka T, Santoro F, Chen D, Gapp BV, Kralovics R, Brummelkamp TR, Nijman SM, Burckstummer T (2014) Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line. Genome Res 24:2059–2065

    Article  CAS  Google Scholar 

  13. Liu SJ, Lim DA (2018) Modulating the expression of long non‐coding RNAs for functional studies. EMBO Rep 19:e46955

    PubMed  PubMed Central  Google Scholar 

  14. Xiang JF, Yin QF, Chen T, Zhang Y, Zhang XO, Wu Z, Zhang S, Wang HB, Ge J, Lu X, Yang L, Chen LL (2014) Human colorectal cancer-specific CCAT1-L lncRNA regulates long-range chromatin interactions at the MYC locus. Cell Res 24:513–531

    Article  CAS  Google Scholar 

  15. Bos TJ, Nussbacher JK, Aigner S, Yeo GW (2016) Tethered function assays as tools to elucidate the molecular roles of RNA-binding proteins. Adv Exp Med Biol 907:61–88

    Article  CAS  Google Scholar 

  16. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308

    Article  CAS  Google Scholar 

  17. Yaguchi K, Yamamoto T, Matsui R, Tsukada Y, Shibanuma A, Kamimura K, Koda T, Uehara R (2018) Uncoordinated centrosome cycle underlies the instability of non-diploid somatic cells in mammals. J Cell Biol 217:2463–2483

    Article  CAS  Google Scholar 

  18. Olbrich T, Mayor-Ruiz C, Vega-Sendino M, Gomez C, Ortega S, Ruiz S, Fernandez-Capetillo O (2017) A p53-dependent response limits the viability of mammalian haploid cells. Proc Natl Acad Sci U S A 114:9367–9372

    Article  CAS  Google Scholar 

  19. Katoh Y, Michisaka S, Nozaki S, Funabashi T, Hirano T, Takei R, Nakayama K (2017) Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated, homology-independent knock-in system. Mol Biol Cell 28:898–906

    Article  CAS  Google Scholar 

  20. Carette JE, Guimaraes CP, Varadarajan M, Park AS, Wuethrich I, Godarova A, Kotecki M, Cochran BH, Spooner E, Ploegh HL, Brummelkamp TR (2009) Haploid genetic screens in human cells identify host factors used by pathogens. Science 326:1231–1235

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T.Y. [26891001, 15K18474, 17K15058, 19H05250, and 19K06479] and T.H. [26113002, 17H03630, 17K19335, 20H00448, 20H05377 and 19K22374]), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to T.Y.), and Tokyo Biochemical Research Foundation (to T.H.).

Author information

Authors and Affiliations

  1. Graduate School of Frontier Biosciences, Osaka University, Suita, Japan

    Tomohiro Yamazaki & Tetsuro Hirose

Authors
  1. Tomohiro Yamazaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tetsuro Hirose
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tetsuro Hirose .

Editor information

Editors and Affiliations

  1. Cardiovascular Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MA, USA

    Haiming Cao

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Cite this protocol

Yamazaki, T., Hirose, T. (2021). CRISPR-Mediated Mutagenesis of Long Noncoding RNAs. In: Cao, H. (eds) Functional Analysis of Long Non-Coding RNAs. Methods in Molecular Biology, vol 2254. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1158-6_18

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1158-6_18

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1157-9

  • Online ISBN: 978-1-0716-1158-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation