Comparison of CRISPR and adenovirus-mediated Myd88 knockdown in RAW 264.7 cells and responses to lipopolysaccharide stimulation


  • Alexander L. Kolb US Army Research Institute of Environmental Medicine
  • Marinaliz Reynoso US Army Research Institute of Environmental Medicine
  • Ronald W. Matheny Military Operational Medicine Research Program



Adenovirus, CRISPR/Cas9, inflammation, MyD88


Genomic manipulation offers the possibility for novel therapies in lieu of medical interventions in use today. The ability to
genetically restore missing inflammatory genes will have a monumental impact on our current immunotherapy treatments. This study compared the efficacy of two different genetic manipulation techniques: clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) transfection to adenoviral transduction to determine which method would provide the most transient and stable knockdown of myeloid differentiation primary response 88 (MyD88). MyD88 is a major regulator of nuclear factor kappa light chain enhancer of activated B cells (NFκB) pathway in Raw 264.7 macrophages. Following genetic manipulation, cells were treated for 24 h with Lipopolysaccharide (LPS) to stimulate the inflammatory pathway. Confirmation of knockdown was determined by western immunoblotting and quantification of band density. Both CRISPR/Cas9 and adenoviral transduction produced similar knockdown efficiency (~64% and 60%, respectively) in MyD88 protein 48 h post adenoviral transduction. NFκB phosphorylation was increased in CRISPR/Cas9-mediated MyD88 knockdown and control cells, but not in adenovirus-mediated MyD88 knockdown cells, following LPS administration. CRISPR/Cas9-mediated MyD88 knockdown macrophages treated with LPS for 24 h showed a 65% reduction in tumor necrosis factor alpha (TNFα) secretion, and a 67% reduction in interleukin-10 (IL-10) secretion when compared to LPS-stimulated control cells (P ≤ 0.01 for both). LPS did not stimulate TNFα or IL-10 secretion in adenovirus-mediated control or MyD88 knockdown cells. These data demonstrate that Raw 264.7 macrophages maintain responsiveness to inflammatory stimuli following CRISPR/Cas9-mediated reductions in MyD88, but not following adenovirus-mediated MyD88 knockdown.


Hoyt JC, Robbins RA. Macrolide antibiotics and pulmonary inflammation. FEMS Microbiol Lett. 2001 Nov;205(1):1–7. PMID:11728708

Melero-Jerez C, Ortega MC, Moliné-Velázquez V, Clemente D. Myeloid derived suppressor cells in inflammatory conditions of the central nervous system. Biochim Biophys Acta. 2016 Mar;1862(3):368–80. PMID:26527182

Yi Y, Noh MJ, Lee KH. Current advances in retroviral gene therapy. Curr Gene Ther. 2011 Jun;11(3):218–28. PMID:21453283

Zhou Y, Boudreau DM, Freedman AN. Trends in the use of aspirin and nonsteroidal anti-inflammatory drugs in the general U.S. population. Pharmacoepidemiol Drug Saf. 2014 Jan;23(1):43–50. PMID:23723142

Coxib, traditional NTC, Bhala N, Emberson J, Merhi A, Abramson S, et al. Vascular and upper gastrointestinal effects of non-steroidal anti-inflammatory drugs: meta-analyses of individual participant data from randomised trials. Lancet. 2013;382(9894):769-79.

Lang FF, Bruner JM, Fuller GN, Aldape K, Prados MD, Chang S, et al. Phase I trial of adenovirus-mediated p53 gene therapy for recurrent glioma: biological and clinical results. J Clin Oncol. 2003 Jul;21(13):2508–18. PMID:12839017

Sakurai F, Mizuguchi H, Yamaguchi T, Hayakawa T. Characterization of in vitro and in vivo gene transfer properties of adenovirus serotype 35 vector. Mol Ther. 2003 Nov;8(5):813–21. PMID:14599815

Wang H, La Russa M, Qi LS. CRISPR/Cas9 in Genome Editing and Beyond. Annu Rev Biochem. 2016 Jun;85(1):227–64. PMID:27145843

Yeh P, Perricaudet M. Advances in adenoviral vectors: from genetic engineering to their biology. FASEB J. 1997 Jul;11(8):615–23. PMID:9240963

Ohashi M, Kanai F, Ueno H, Tanaka T, Tateishi K, Kawakami T, et al. Adenovirus mediated p53 tumour suppressor gene therapy for human gastric cancer cells in vitro and in vivo. Gut. 1999 Mar;44(3):366–71. PMID:10026322

Ryuke Y, Mizuno M, Natsume A, Yoshida J. Transduction efficiency of adenoviral vectors into human glioma cells increased by association with cationic liposomes. Neurol Med Chir (Tokyo). 2000 May;40(5):256–60. PMID:11980090

Liao HK, Gu Y, Diaz A, Marlett J, Takahashi Y, Li M, et al. Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells. Nat Commun. 2015 Mar;6(1):6413. PMID:25752527

Guo R, Wan Y, Xu D, Cui L, Deng M, Zhang G, et al. Generation and evaluation of Myostatin knock-out rabbits and goats using CRISPR/Cas9 system. Sci Rep. 2016 Jul;6(1):29855. PMID:27417210

Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013 Nov;8(11):2281–308. PMID:24157548

Zhang XH, Tee LY, Wang XG, Huang QS, Yang SH. Off-target Effects in CRISPR/Cas9-mediated Genome Engineering. Mol Ther Nucleic Acids. 2015 Nov;4:e264. PMID:26575098

Lau CH, Suh Y. In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease. F1000 Res. 2017 Dec;6:2153. PMID:29333255

Dow LE, Fisher J, O’Rourke KP, Muley A, Kastenhuber ER, Livshits G, et al. Inducible in vivo genome editing with CRISPR-Cas9. Nat Biotechnol. 2015 Apr;33(4):390–4. PMID:25690852

Lawrence T. The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb Perspect Biol. 2009 Dec;1(6):a001651. PMID:20457564

Wang JQ, Jeelall YS, Ferguson LL, Horikawa K. Toll-Like Receptors and Cancer: MYD88 Mutation and Inflammation. Front Immunol. 2014 Jul;5:367. PMID:25132836

Hobbs S, Reynoso M, Geddis AV, Mitrophanov AY, Matheny RW Jr. LPS-stimulated NF-κB p65 dynamic response marks the initiation of TNF expression and transition to IL-10 expression in RAW 264.7 macrophages. Physiol Rep. 2018 Nov;6(21):e13914. PMID:30426723

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May;72(1-2):248–54. PMID:942051

Matheny RW Jr, Lynch CM, Leandry LA. Enhanced Akt phosphorylation and myogenic differentiation in PI3K p110β-deficient myoblasts is mediated by PI3K p110α and mTORC2. Growth Factors. 2012 Dec;30(6):367–84. PMID:23137199

Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012 Jul;9(7):671–5. PMID:22930834

Yamada R, Ymamoto K. Recent findings on genes associated with inflammatory disease. Mutat Res. 2005 Jun;573(1-2):136–51. PMID:15829243

Tuppen HA, Blakely EL, Turnbull DM, Taylor RW. Mitochondrial DNA mutations and human disease. Biochim Biophys Acta. 2010 Feb;1797(2):113–28. PMID:19761752

Wilmut I, Hooper ML, Simons JP. Genetic manipulation of mammals and its application in reproductive biology. J Reprod Fertil. 1991 Jul;92(2):245–79. PMID:1886087

van der Weyden L, Adams DJ, Bradley A. Tools for targeted manipulation of the mouse genome. Physiol Genomics. 2002 Dec;11(3):133–64. PMID:12464689

Corridon PR, Rhodes GJ, Leonard EC, Basile DP, Gattone VH 2nd, Bacallao RL, et al. A method to facilitate and monitor expression of exogenous genes in the rat kidney using plasmid and viral vectors. Am J Physiol Renal Physiol. 2013 May;304(9):F1217–29. PMID:23467422

Veach RA, Wilson MH. CRISPR/Cas9 engineering of a KIM-1 reporter human proximal tubule cell line. PLoS One. 2018 Sep;13(9):e0204487. PMID:30260998

Matheny RW Jr, Riddle-Kottke MA, Leandry LA, Lynch CM, Abdalla MN, Geddis AV, et al. Role of phosphoinositide 3-OH kinase p110β in skeletal myogenesis. Mol Cell Biol. 2015 Apr;35(7):1182–96. PMID:25605332

graphical abstract


Additional Files



How to Cite

Kolb AL, Reynoso M, Matheny RW. Comparison of CRISPR and adenovirus-mediated Myd88 knockdown in RAW 264.7 cells and responses to lipopolysaccharide stimulation. J Biol Methods [Internet]. 2021Jul.15 [cited 2021Dec.4];8(3):e151. Available from: