Skip to main content
SpringerLink
Log in

Functional Analysis of Variants in BRCA1 Using CRISPR Base Editors

  • Protocol
  • First Online:
Base Editors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2606))

Abstract

To date, methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays have been used to evaluate the function of the variants of uncertain significance (VUS) of the BRCA genes. However, these methods have limitations as they are associated with overexpression and do not apply to post-transcriptional regulation. Therefore, there are several VUS whose functions are unclear. Recently, we devised a new way to assess the functionality of variants in BRCA1 via a CRISPR-mediated base editor to overcome these limitations. We precisely introduced the target nucleotide substitution in living cells and identified variants whose functions were not defined. Here, we describe the methods for the functional appraisal of BRCA1 variants using CRISPR-based base editors.

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 219.99
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. Roy R, Chun J, Powell SN (2011) BRCA1 and BRCA2: different roles in a common pathway of genome protection. Nat Rev Cancer 12(1):68–78. https://doi.org/10.1038/nrc3181

    Article  CAS  Google Scholar 

  2. Kuchenbaecker KB, Hopper JL, Barnes DR, Phillips KA, Mooij TM, Roos-Blom MJ, Jervis S, van Leeuwen FE, Milne RL, Andrieu N, Goldgar DE, Terry MB, Rookus MA, Easton DF, Antoniou AC, BRCA1 and BRCA2 Cohort Consortium, McGuffog L, Evans DG, Barrowdale D, Frost D, Adlard J, Ong KR, Izatt L, Tischkowitz M, Eeles R, Davidson R, Hodgson S, Ellis S, Nogues C, Lasset C, Stoppa-Lyonnet D, Fricker JP, Faivre L, Berthet P, Hooning MJ, van der Kolk LE, Kets CM, Adank MA, John EM, Chung WK, Andrulis IL, Southey M, Daly MB, Buys SS, Osorio A, Engel C, Kast K, Schmutzler RK, Caldes T, Jakubowska A, Simard J, Friedlander ML, McLachlan SA, Machackova E, Foretova L, Tan YY, Singer CF, Olah E, Gerdes AM, Arver B, Olsson H (2017) Risks of breast, ovarian, and contralateral breast cancer for BRCA1 and BRCA2 mutation carriers. JAMA 317(23):2402–2416. https://doi.org/10.1001/jama.2017.7112

    Article  CAS  Google Scholar 

  3. Millot GA, Carvalho MA, Caputo SM, Vreeswijk MP, Brown MA, Webb M, Rouleau E, Neuhausen SL, Hansen T, Galli A, Brandao RD, Blok MJ, Velkova A, Couch FJ, Monteiro AN, Group ECFAW (2012) A guide for functional analysis of BRCA1 variants of uncertain significance. Hum Mutat 33(11):1526–1537. https://doi.org/10.1002/humu.22150

    Article  CAS  Google Scholar 

  4. Santos C, Peixoto A, Rocha P, Pinto P, Bizarro S, Pinheiro M, Pinto C, Henrique R, Teixeira MR (2014) Pathogenicity evaluation of BRCA1 and BRCA2 unclassified variants identified in Portuguese breast/ovarian cancer families. J Mol Diagn 16(3):324–334. https://doi.org/10.1016/j.jmoldx.2014.01.005

    Article  CAS  Google Scholar 

  5. Starita LM, Islam MM, Banerjee T, Adamovich AI, Gullingsrud J, Fields S, Shendure J, Parvin JD (2018) A multiplex homology-directed DNA repair assay reveals the impact of more than 1,000 BRCA1 missense substitution variants on protein function. Am J Hum Genet 103(4):498–508. https://doi.org/10.1016/j.ajhg.2018.07.016

    Article  CAS  Google Scholar 

  6. Anantha RW, Simhadri S, Foo TK, Miao S, Liu J, Shen Z, Ganesan S, Xia B (2017) Functional and mutational landscapes of BRCA1 for homology-directed repair and therapy resistance. elife 6. https://doi.org/10.7554/eLife.21350

  7. Quann K, Jing Y, Rigoutsos I (2015) Post-transcriptional regulation of BRCA1 through its coding sequence by the miR-15/107 group of miRNAs. Front Genet 6:242. https://doi.org/10.3389/fgene.2015.00242

    Article  CAS  Google Scholar 

  8. Saunus JM, French JD, Edwards SL, Beveridge DJ, Hatchell EC, Wagner SA, Stein SR, Davidson A, Simpson KJ, Francis GD, Leedman PJ, Brown MA (2008) Posttranscriptional regulation of the breast cancer susceptibility gene BRCA1 by the RNA binding protein HuR. Cancer Res 68(22):9469–9478. https://doi.org/10.1158/0008-5472.CAN-08-1159

    Article  CAS  Google Scholar 

  9. Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32(4):347–355. https://doi.org/10.1038/nbt.2842

    Article  CAS  Google Scholar 

  10. Hess GT, Fresard L, Han K, Lee CH, Li A, Cimprich KA, Montgomery SB, Bassik MC (2016) Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat Methods 13(12):1036–1042. https://doi.org/10.1038/nmeth.4038

    Article  CAS  Google Scholar 

  11. Kim K, Ryu SM, Kim ST, Baek G, Kim D, Lim K, Chung E, Kim S, Kim JS (2017) Highly efficient RNA-guided base editing in mouse embryos. Nat Biotechnol 35(5):435–437. https://doi.org/10.1038/nbt.3816

    Article  CAS  Google Scholar 

  12. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533(7603):420–424. https://doi.org/10.1038/nature17946

    Article  CAS  Google Scholar 

  13. Park DS, Yoon M, Kweon J, Jang AH, Kim Y, Choi SC (2017) Targeted Base editing via RNA-guided cytidine deaminases in Xenopus laevis embryos. Mol Cells 40(11):823–827. https://doi.org/10.14348/molcells.2017.0262

    Article  CAS  Google Scholar 

  14. Walton RT, Christie KA, Whittaker MN, Kleinstiver BP (2020) Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 368:290–296. https://doi.org/10.1126/science.aba8853

    Article  CAS  Google Scholar 

  15. Rees HA, Komor AC, Yeh WH, Caetano-Lopes J, Warman M, Edge ASB, Liu DR (2017) Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat Commun 8:15790. https://doi.org/10.1038/ncomms15790

    Article  CAS  Google Scholar 

  16. Koblan LW, Doman JL, Wilson C, Levy JM, Tay T, Newby GA, Maianti JP, Raguram A, Liu DR (2018) Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol 36(9):843–846. https://doi.org/10.1038/nbt.4172

    Article  CAS  Google Scholar 

  17. Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR (2017) Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551(7681):464–471. https://doi.org/10.1038/nature24644

    Article  CAS  Google Scholar 

  18. Kweon J, Jang AH, Shin HR, See JE, Lee W, Lee JW, Chang S, Kim K, Kim Y (2020) A CRISPR-based base-editing screen for the functional assessment of BRCA1 variants. Oncogene 39(1):30–35. https://doi.org/10.1038/s41388-019-0968-2

    Article  CAS  Google Scholar 

  19. See JE, Shin HR, Jang G, Kweon J, Kim Y (2021) Functional assessment of BRCA1 variants using CRISPR-mediated base editors. J Vis Exp 168. https://doi.org/10.3791/61557

  20. Gibson DG (2011) Enzymatic assembly of overlapping DNA fragments. Methods Enzymol 498:349–361. https://doi.org/10.1016/B978-0-12-385120-8.00015-2

    Article  CAS  Google Scholar 

  21. Nageshwaran S, Chavez A, Cher Yeo N, Guo X, Lance-Byrne A, Tung A, Collins JJ, Church GM (2018) CRISPR guide RNA cloning for mammalian systems. J Vis Exp 140. https://doi.org/10.3791/57998

  22. Kweon J, Kim DE, Jang AH, Kim Y (2018) CRISPR/Cas-based customization of pooled CRISPR libraries. PLoS One 13(6):e0199473. https://doi.org/10.1371/journal.pone.0199473

    Article  CAS  Google Scholar 

  23. Kim Y, Kweon J, Kim A, Chon JK, Yoo JY, Kim HJ, Kim S, Lee C, Jeong E, Chung E, Kim D, Lee MS, Go EM, Song HJ, Kim H, Cho N, Bang D, Kim S, Kim JS (2013) A library of TAL effector nucleases spanning the human genome. Nat Biotechnol 31(3):251–258. https://doi.org/10.1038/nbt.2517

    Article  CAS  Google Scholar 

  24. Kim D, Kim DE, Lee G, Cho SI, Kim JS (2019) Genome-wide target specificity of CRISPR RNA-guided adenine base editors. Nat Biotechnol 37(4):430–435. https://doi.org/10.1038/s41587-019-0050-1

    Article  CAS  Google Scholar 

  25. Clement K (2019) CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol 37(3):224–226. https://doi.org/10.1038/s41587-019-0043-0

    Article  CAS  Google Scholar 

  26. Hwang GH, Park J, Lim K, Kim S, Yu J, Yu E, Kim ST, Eils R, Kim JS, Bae S (2018) Web-based design and analysis tools for CRISPR base editing. BMC Bioinformatics 19(1):542. https://doi.org/10.1186/s12859-018-2585-4

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported by the National Research Foundation of Korea (grants 2017M3A9B4062419, 2020R1F1A1075508, 2018R1A5A2020732, and 2021R1C1C1007162 to YK and 2021R1A6A3A13046696 to J.-E.S.).

Author information

Authors and Affiliations

  1. Department of Biomedical Sciences, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea

    Ji-Eun See & Yongsub Kim

  2. Stem Cell Immunomodulation Research Center, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea

    Ji-Eun See & Yongsub Kim

Authors
  1. Ji-Eun See
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yongsub Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Seoul National University College of Medicine, Seoul, Korea (Republic of)

    Sangsu Bae

  2. Seoul National University College of Medicine, Seoul, Korea (Republic of)

    Beomjong Song

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

See, JE., Kim, Y. (2023). Functional Analysis of Variants in BRCA1 Using CRISPR Base Editors. In: Bae, S., Song, B. (eds) Base Editors. Methods in Molecular Biology, vol 2606. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2879-9_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2879-9_7

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2878-2

  • Online ISBN: 978-1-0716-2879-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation