Primary Airway Epithelial Cell Gene Editing Using CRISPR-Cas9

  • Jamie L. EvermanEmail author
  • Cydney Rios
  • Max A. Seibold
Part of the Methods in Molecular Biology book series (MIMB, volume 1706)


The adaptation of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated endonuclease 9 (CRISPR-Cas9) machinery from prokaryotic organisms has resulted in a gene editing system that is highly versatile, easily constructed, and can be leveraged to generate human cells knocked out (KO) for a specific gene. While standard transfection techniques can be used for the introduction of CRISPR-Cas9 expression cassettes to many cell types, delivery by this method is not efficient in many primary cell types, including primary human airway epithelial cells (AECs). More efficient delivery in AECs can be achieved through lentiviral-mediated transduction, allowing the CRISPR-Cas9 system to be integrated into the genome of the cell, resulting in stable expression of the nuclease machinery and increasing editing rates. In parallel, advancements have been made in the culture, expansion, selection, and differentiation of AECs, which allow the robust generation of a bulk edited AEC population from transduced cells. Applying these methods, we detail here our latest protocol to generate mucociliary epithelial cultures knocked out for a specific gene from donor-isolated primary human basal airway epithelial cells. This protocol includes methods to: (1) design and generate lentivirus which targets a specific gene for KO with CRISPR-Cas9 machinery, (2) efficiently transduce AECs, (3) culture and select for a bulk edited AEC population, (4) molecularly screen AECs for Cas9 cutting and specific sequence edits, and (5) further expand and differentiate edited cells to a mucociliary airway epithelial culture. The AEC knockouts generated using this protocol provide an excellent primary cell model system with which to characterize the function of genes involved in airway dysfunction and disease.

Key words

CRISPR Gene editing Lentivirus Airway epithelial cells Primary cells Gene knockout 


  1. 1.
    Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):806–811. CrossRefPubMedGoogle Scholar
  2. 2.
    Ramachandran S, Krishnamurthy S, Jacobi AM, Wohlford-Lenane C, Behlke MA, Davidson BL, PB MC Jr (2013) Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro. Am J Physiol Lung Cell Mol Physiol 305(1):L23–L32. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Hammond SM (2005) Dicing and slicing: the core machinery of the RNA interference pathway. FEBS Lett 579(26):5822–5829. CrossRefPubMedGoogle Scholar
  4. 4.
    Tomari Y, Zamore PD (2005) Perspective: machines for RNAi. Genes Dev 19(5):517–529. CrossRefPubMedGoogle Scholar
  5. 5.
    Mocellin S, Provenzano M (2004) RNA interference: learning gene knock-down from cell physiology. J Transl Med 2(1):39. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326(5959):1509–1512. CrossRefPubMedGoogle Scholar
  7. 7.
    Hurt JA, Thibodeau SA, Hirsh AS, Pabo CO, Joung JK (2003) Highly specific zinc finger proteins obtained by directed domain shuffling and cell-based selection. Proc Natl Acad Sci U S A 100(21):12271–12276. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Wright DA, Thibodeau-Beganny S, Sander JD, Winfrey RJ, Hirsh AS, Eichtinger M, Fu F, Porteus MH, Dobbs D, Voytas DF, Joung JK (2006) Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nat Protoc 1(3):1637–1652. CrossRefPubMedGoogle Scholar
  9. 9.
    Zhang F, Cong L, Lodato S, Kosuri S, Church GM, Arlotta P (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol 29(2):149–153. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, Soria E (2005) Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 60(2):174–182. CrossRefPubMedGoogle Scholar
  11. 11.
    Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315(5819):1709–1712. CrossRefPubMedGoogle Scholar
  12. 12.
    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    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(6096):816–821. CrossRefPubMedGoogle Scholar
  14. 14.
    Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471(7340):602–607. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Karvelis T, Gasiunas G, Miksys A, Barrangou R, Horvath P, Siksnys V (2013) crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus Thermophilus. RNA Biol 10(5):841–851. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321(5891):960–964. CrossRefPubMedGoogle Scholar
  17. 17.
    Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281–2308. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343(6166):84–87. CrossRefPubMedGoogle Scholar
  19. 19.
    Sanjana NE, Shalem O, Zhang F (2014) Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11(8):783–784. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Chu HW, Rios C, Huang C, Wesolowska-Andersen A, Burchard EG, O'Connor BP, Fingerlin TE, Nichols D, Reynolds SD, Seibold MA (2015) CRISPR-Cas9-mediated gene knockout in primary human airway epithelial cells reveals a proinflammatory role for MUC18. Gene Ther 22(10):822–829. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Bellec J, Bacchetta M, Losa D, Anegon I, Chanson M, Nguyen TH (2015) CFTR inactivation by lentiviral vector-mediated RNA interference and CRISPR-Cas9 genome editing in human airway epithelial cells. Curr Gene Ther 15(5):447–459CrossRefPubMedGoogle Scholar
  22. 22.
    Firth AL, Menon T, Parker GS, Qualls SJ, Lewis BM, Ke E, Dargitz CT, Wright R, Khanna A, Gage FH, Verma IM (2015) Functional gene correction for cystic fibrosis in lung epithelial cells generated from patient iPSCs. Cell Rep 12(9):1385–1390. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Reynolds SD, Rios C, Wesolowska-Andersen A, Zhuang Y, Pinter M, Happoldt C, Hill CL, Lallier SW, Cosgrove GP, Solomon GM, Nichols DP, Seibold MA (2016) Airway progenitor clone formation is enhanced by Y-27632-dependent changes in the Transcriptome. Am J Respir Cell Mol Biol 55(3):323–336. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    SAM target sgRNA cloning protocol (2014)
  25. 25.
    Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, EY Y, An DS, Sabatini DM, Chen IS, Hahn WC, Sharp PA, Weinberg RA, Novina CD (2003) Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 9(4):493–501CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Bolotin A, Quinquis B, Sorokin A, Ehrlich SD (2005) Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151(Pt 8):2551–2561. PubMedGoogle Scholar
  27. 27.
    Suprynowicz FA, Upadhyay G, Krawczyk E, Kramer SC, Hebert JD, Liu X, Yuan H, Cheluvaraju C, Clapp PW, Boucher RC Jr, Kamonjoh CM, Randell SH, Schlegel R (2012) Conditionally reprogrammed cells represent a stem-like state of adult epithelial cells. Proc Natl Acad Sci U S A 109(49):20035–20040. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Liu X, Ory V, Chapman S, Yuan H, Albanese C, Kallakury B, Timofeeva OA, Nealon C, Dakic A, Simic V, Haddad BR, Rhim JS, Dritschilo A, Riegel A, McBride A, Schlegel R (2012) ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. Am J Pathol 180(2):599–607. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Jamie L. Everman
    • 1
    Email author
  • Cydney Rios
    • 1
  • Max A. Seibold
    • 1
    • 2
  1. 1.Center for Genes, Environment, and HealthNational Jewish HealthDenverUSA
  2. 2.Department of PediatricsNational Jewish HealthDenverUSA

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