#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Off-target effects of CRISPRa on interleukin-6 expression


Autoři: Sébastien Soubeyrand aff001;  Paulina Lau aff001;  Victoria Peters aff001;  Ruth McPherson aff001
Působiště autorů: Atherogenomics Laboratory, University of Ottawa Heart Institute, Ottawa, Ontario, Canada aff001;  Department of Medicine, Ruddy Canadian Cardiovascular Genetics Centre, University of Ottawa Heart Institute, Ottawa, Ontario, Canada aff002
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0224113

Souhrn

Inactive fusion variants of the CRISPR-Cas9 system are increasingly being used as standard methodology to study transcription regulation. Their ability to readily manipulate the native genomic loci is particularly advantageous. In this work, we serendipitously uncover the key cytokine IL6 as an off-target of the activating derivative of CRISPR (CRISPRa) while studying RP11-326A19.4, a novel long-non coding RNA (lncRNA). Increasing RP11-326A19.4 expression in HEK293T cells via CRISPRa-mediated activation of its promoter region induced genome-wide transcriptional changes, including upregulation of IL6, an important cytokine. IL6 was increased in response to distinct sgRNA targeting the RP11-326A19.4 promoter region, suggesting specificity. Loss of the cognate sgRNA recognition sites failed to abolish CRISPRa mediated activation of IL6 however, pointing to off-target effects. Bioinformatic approaches did not reveal predicted off-target binding sites. Off-target activation of IL6 was sustained and involved low level activation of known IL6 regulators. Increased IL6 remained sensitive to further activation by TNFα, consistent with the existence of independent mechanisms. This study provides experimental evidence that CRISPRa has discrete, unpredictable off-targeting limitations that must be considered when using this emerging technology.

Klíčová slova:

CRISPR – DNA transcription – Genetic loci – Long non-coding RNAs – Promoter regions – Regulator genes – Transcriptional control – Transfection


Zdroje

1. Xu X, Gao J, Dai W, Wang D, Wu J, Wang J. Gene activation by a CRISPR-assisted trans enhancer. Elife. 2019;8.

2. Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, P R Iyer E, et al. Highly efficient Cas9-mediated transcriptional programming. Nat Methods. NIH Public Access; 2015;12: 326–8.

3. Jiang F, Doudna JA. CRISPR–Cas9 Structures and Mechanisms. Annu Rev Biophys. Annual Reviews; 2017;46: 505–529.

4. Aryal NK, Wasylishen AR, Lozano G. CRISPR/Cas9 can mediate high-efficiency off-target mutations in mice in vivo. Cell Death Dis. Nature Publishing Group; 2018;9: 1099.

5. Wu X, Scott DA, Kriz AJ, Chiu AC, Hsu PD, Dadon DB, et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat Biotechnol. NIH Public Access; 2014;32: 670–6.

6. O’Geen H, Henry IM, Bhakta MS, Meckler JF, Segal DJ. A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture. Nucleic Acids Res. Oxford University Press; 2015;43: 3389–404.

7. Horlbeck MA, Witkowsky LB, Guglielmi B, Replogle JM, Gilbert LA, Villalta JE, et al. Nucleosomes impede Cas9 access to DNA in vivo and in vitro. Elife. 2016;5.

8. Deveson IW, Brunck ME, Blackburn J, Tseng E, Hon T, Clark TA, et al. Universal Alternative Splicing of Noncoding Exons. Cell Syst. 2018;6: 245–255.e5. doi: 10.1016/j.cels.2017.12.005 29396323

9. Morris K V, Mattick JS. The rise of regulatory RNA. NatRevGenet. 2014;15: 423–437.

10. Ulitsky I, Bartel DP. lincRNAs: genomics, evolution, and mechanisms. Cell. 2013;154: 26–46. doi: 10.1016/j.cell.2013.06.020 23827673

11. Liu SJ, Horlbeck MA, Cho SW, Birk HS, Malatesta M, He D, et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science (80-). NIH Public Access; 2017;355.

12. Joung J, Engreitz JM, Konermann S, Abudayyeh OO, Verdine VK, Aguet F, et al. Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood. Nature. Nature Publishing Group; 2017;548: 343–346.

13. Paralkar VR, Taborda CC, Huang P, Yao Y, Kossenkov AV, Prasad R, et al. Unlinking an lncRNA from Its Associated cis Element. Mol Cell. Cell Press; 2016;62: 104–110.

14. Scheller J, Chalaris A, Schmidt-Arras D, Rose-John S. The pro- and anti-inflammatory properties of the cytokine interleukin-6. Biochim Biophys Acta—Mol Cell Res. Elsevier; 2011;1813: 878–888.

15. Tanaka T, Narazaki M, Kishimoto T. IL-6 in inflammation, immunity, and disease. Cold Spring Harb Perspect Biol. Cold Spring Harbor Laboratory Press; 2014;6: a016295.

16. Lee JW, Stone ML, Porrett PM, Thomas SK, Komar CA, Li JH, et al. Hepatocytes direct the formation of a pro-metastatic niche in the liver. Nature. Nature Publishing Group; 2019;567: 249–252.

17. Deloukas P, Kanoni S, Willenborg C, Farrall M, Assimes TL, Thompson JR, et al. Large-scale association analysis identifies new risk loci for coronary artery disease. Nat Genet. 2013;45: 25–33. doi: 10.1038/ng.2480 23202125

18. Soubeyrand S, Nikpay M, Turner A, Dang A-T, Herfkens M, Lau P, et al. Regulation of MFGE8 by the intergenic coronary artery disease locus on 15q26.1. Atherosclerosis. 2019;284: 11–17. doi: 10.1016/j.atherosclerosis.2019.02.012 30861420

19. Stemmer M, Thumberger T, del Sol Keyer M, Wittbrodt J, Mateo JL. CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool. Maas S, editor. PLoS One. Public Library of Science; 2015;10: e0124633.

20. Moreno-Mateos MA, Vejnar CE, Beaudoin J-D, Fernandez JP, Mis EK, Khokha MK, et al. CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat Methods. 2015;12: 982–8. doi: 10.1038/nmeth.3543 26322839

21. Xu H, Xiao T, Chen C-H, Li W, Meyer CA, Wu Q, et al. Sequence determinants of improved CRISPR sgRNA design. Genome Res. 2015;25: 1147–1157. doi: 10.1101/gr.191452.115 26063738

22. Liu H, Wei Z, Dominguez A, Li Y, Wang X, Qi LS. CRISPR-ERA: a comprehensive design tool for CRISPR-mediated gene editing, repression and activation. Bioinformatics. 2015;31: 3676–8. doi: 10.1093/bioinformatics/btv423 26209430

23. Singh R, Kuscu C, Quinlan A, Qi Y, Adli M. Cas9-chromatin binding information enables more accurate CRISPR off-target prediction. Nucleic Acids Res. Narnia; 2015;43: e118–e118.

24. Lin Y, Cradick TJ, Brown MT, Deshmukh H, Ranjan P, Sarode N, et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res. Oxford University Press; 2014;42: 7473–85.

25. Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, et al. The Sequence of the Human Genome. Science (80-). 2001;291: 1304–1351.

26. Kojima Y, Volkmer J-P, McKenna K, Civelek M, Lusis AJ, Miller CL, et al. CD47-blocking antibodies restore phagocytosis and prevent atherosclerosis. Nature. 2016;536: 86–90. doi: 10.1038/nature18935 27437576

27. Schmidt-Arras D, Rose-John S. IL-6 pathway in the liver: From physiopathology to therapy. J Hepatol. 2016;64: 1403–1415. doi: 10.1016/j.jhep.2016.02.004 26867490

28. Engreitz JM, Haines JE, Perez EM, Munson G, Chen J, Kane M, et al. Local regulation of gene expression by lncRNA promoters, transcription and splicing. Nature. 2016;539: 452–455. doi: 10.1038/nature20149 27783602

29. Hao S, Baltimore D. The stability of mRNA influences the temporal order of the induction of genes encoding inflammatory molecules. Nat Immunol. NIH Public Access; 2009;10: 281–8.

30. Schoenfelder S, Fraser P. Long-range enhancer–promoter contacts in gene expression control. Nat Rev Genet. Nature Publishing Group; 2019; 1.

31. Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. NIH Public Access; 2014;32: 279–284.

32. Amrani N, Gao XD, Liu P, Edraki A, Mir A, Ibraheim R, et al. NmeCas9 is an intrinsically high-fidelity genome-editing platform. Genome Biol. BioMed Central; 2018;19: 214.

33. Yourik P, Fuchs RT, Mabuchi M, Curcuru JL, Robb GB. Staphylococcus aureus Cas9 is a multiple-turnover enzyme. RNA. 2019;25: 35–44. doi: 10.1261/rna.067355.118 30348755

34. Nikpay M, Goel A, Won H-H, Hall LM, Willenborg C, Kanoni S, et al. A comprehensive 1000 Genomes-based genome-wide association meta-analysis of coronary artery disease. Nat Genet. 2015;47: 1121–30. doi: 10.1038/ng.3396 26343387


Článek vyšel v časopise

PLOS One


2019 Číslo 10
Nejčtenější tento týden
Nejčtenější v tomto čísle
Kurzy

Zvyšte si kvalifikaci online z pohodlí domova

KOST
Koncepce osteologické péče pro gynekology a praktické lékaře
nový kurz
Autoři: MUDr. František Šenk

Sekvenční léčba schizofrenie
Autoři: MUDr. Jana Hořínková

Hypertenze a hypercholesterolémie – synergický efekt léčby
Autoři: prof. MUDr. Hana Rosolová, DrSc.

Svět praktické medicíny 5/2023 (znalostní test z časopisu)

Imunopatologie? … a co my s tím???
Autoři: doc. MUDr. Helena Lahoda Brodská, Ph.D.

Všechny kurzy
Kurzy Podcasty Doporučená témata Časopisy
Přihlášení
Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.

Přihlášení

Nemáte účet?  Registrujte se

#ADS_BOTTOM_SCRIPTS#