Abstract
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
This is a preview of subscription content, log in via an institution.
References
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. https://doi.org/10.1038/35888
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. https://doi.org/10.1152/ajplung.00426.2012
Hammond SM (2005) Dicing and slicing: the core machinery of the RNA interference pathway. FEBS Lett 579(26):5822–5829. https://doi.org/10.1016/j.febslet.2005.08.079
Tomari Y, Zamore PD (2005) Perspective: machines for RNAi. Genes Dev 19(5):517–529. https://doi.org/10.1101/gad.1284105
Mocellin S, Provenzano M (2004) RNA interference: learning gene knock-down from cell physiology. J Transl Med 2(1):39. https://doi.org/10.1186/1479-5876-2-39
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. https://doi.org/10.1126/science.1178811
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. https://doi.org/10.1073/pnas.2135381100
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. https://doi.org/10.1038/nprot.2006.259
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. https://doi.org/10.1038/nbt.1775
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. https://doi.org/10.1007/s00239-004-0046-3
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. https://doi.org/10.1126/science.1138140
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. https://doi.org/10.1126/science.1231143
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. https://doi.org/10.1126/science.1225829
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. https://doi.org/10.1038/nature09886
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. https://doi.org/10.4161/rna.24203
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. https://doi.org/10.1126/science.1159689
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. https://doi.org/10.1038/nprot.2013.143
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. https://doi.org/10.1126/science.1247005
Sanjana NE, Shalem O, Zhang F (2014) Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11(8):783–784. https://doi.org/10.1038/nmeth.3047
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. https://doi.org/10.1038/gt.2015.53
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):447–459
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. https://doi.org/10.1016/j.celrep.2015.07.062
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. https://doi.org/10.1165/rcmb.2015-0274MA
SAM target sgRNA cloning protocol (2014) http://sam.genome-engineering.org/protocols/
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):493–501
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. https://doi.org/10.1099/mic.0.28048-0
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. https://doi.org/10.1073/pnas.1213241109
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. https://doi.org/10.1016/j.ajpath.2011.10.036
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Everman, J.L., Rios, C., Seibold, M.A. (2018). Primary Airway Epithelial Cell Gene Editing Using CRISPR-Cas9. In: DiStefano, J. (eds) Disease Gene Identification. Methods in Molecular Biology, vol 1706. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7471-9_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7471-9_15
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7470-2
Online ISBN: 978-1-4939-7471-9
eBook Packages: Springer Protocols