دورية أكاديمية
Robust and heritable knockdown of gene expression using a self-cleaving ribozyme in Drosophila.
| العنوان: | Robust and heritable knockdown of gene expression using a self-cleaving ribozyme in Drosophila. |
|---|---|
| المؤلفون: | Nyberg KG; Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA., Navales FG; Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA., Keles E; Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA., Nguyen JQ; Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA., Hertz LM; Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA., Carthew RW; Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA.; NSF-Simons National Institute for Theory and Mathematics in Biology, Evanston, IL 60208, USA.; NSF-Simons Center for Quantitative Biology, Evanston, IL 60208, USA. |
| المصدر: | Genetics [Genetics] 2024 Aug 07; Vol. 227 (4). |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: Oxford University Press Country of Publication: United States NLM ID: 0374636 Publication Model: Print Cited Medium: Internet ISSN: 1943-2631 (Electronic) Linking ISSN: 00166731 NLM ISO Abbreviation: Genetics Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: 2021- : [Oxford] : Oxford University Press Original Publication: Austin, Tex. [etc.] |
| مواضيع طبية MeSH: | RNA, Catalytic*/genetics , RNA, Catalytic*/metabolism , Drosophila melanogaster*/genetics , Gene Knockdown Techniques*, Animals ; CRISPR-Cas Systems ; Male |
| مستخلص: | The current toolkit for genetic manipulation in the model animal Drosophila melanogaster is extensive and versatile but not without its limitations. Here, we report a powerful and heritable method to knockdown gene expression in D. melanogaster using the self-cleaving N79 hammerhead ribozyme, a modification of a naturally occurring ribozyme found in the parasite Schistosoma mansoni. A 111-bp ribozyme cassette, consisting of the N79 ribozyme surrounded by insulating spacer sequences, was inserted into 4 independent long noncoding RNA genes as well as the male-specific splice variant of doublesex using scarless CRISPR/Cas9-mediated genome editing. Ribozyme-induced RNA cleavage resulted in robust destruction of 3' fragments typically exceeding 90%. Single molecule RNA fluorescence in situ hybridization results suggest that cleavage and destruction can even occur for nascent transcribing RNAs. Knockdown was highly specific to the targeted RNA, with no adverse effects observed in neighboring genes or the other splice variants. To control for potential effects produced by the simple insertion of 111 nucleotides into genes, we tested multiple catalytically inactive ribozyme variants and found that a variant with scrambled N79 sequence best recapitulated natural RNA levels. Thus, self-cleaving ribozymes offer a novel approach for powerful gene knockdown in Drosophila, with potential applications for the study of noncoding RNAs, nuclear-localized RNAs, and specific splice variants of protein-coding genes. Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest. (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.) |
| References: | Cell. 2009 Feb 20;136(4):642-55. (PMID: 19239886) Genes Dev. 1988 Apr;2(4):477-89. (PMID: 2453394) Life Sci Alliance. 2018 Jul 31;1(4):e201800124. (PMID: 30456373) Nat Methods. 2006 Oct;3(10):833-8. (PMID: 16964256) Commun Biol. 2023 Aug 4;6(1):816. (PMID: 37542105) Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11157-61. (PMID: 8248222) Methods Mol Biol. 2022;2540:135-156. (PMID: 35980576) Genes Dev. 1988 Apr;2(4):440-52. (PMID: 2836265) Cancers (Basel). 2020 Jun 03;12(6):. (PMID: 32503290) Algorithms Mol Biol. 2011 Nov 24;6:26. (PMID: 22115189) Genetics. 2013 Aug;194(4):1029-35. (PMID: 23709638) PLoS One. 2020 Apr 30;15(4):e0232046. (PMID: 32352996) Methods Mol Biol. 2022;2540:1-34. (PMID: 35980571) Nat Rev Genet. 2007 Oct;8(10):776-90. (PMID: 17846637) Science. 2000 Jun 16;288(5473):2013-8. (PMID: 10856208) Fly (Austin). 2020 Mar - Dec;14(1-4):34-48. (PMID: 33016195) PLoS One. 2012;7(2):e30619. (PMID: 22312429) Cell. 1997 Feb 21;88(4):445-57. (PMID: 9038336) J Cell Biol. 2017 Jan 2;216(1):231-246. (PMID: 27998991) J Cell Sci. 2015 Feb 1;128(3):499-515. (PMID: 25413347) Cell. 1997 Feb 21;88(4):459-69. (PMID: 9038337) Mol Cell Biol. 2011 Feb;31(4):639-51. (PMID: 21135120) Genetics. 1975 Aug;80(4):733-52. (PMID: 811500) Development. 2001 Apr;128(7):1033-43. (PMID: 11245569) Mol Cell Biol. 1998 Jul;18(7):3880-8. (PMID: 9632772) Bioinformatics. 2014 Aug 1;30(15):2114-20. (PMID: 24695404) Nat Biotechnol. 2016 May;34(5):525-7. (PMID: 27043002) PLoS Biol. 2010 May 04;8(5):e1000365. (PMID: 20454565) Bioinformatics. 2011 Jul 1;27(13):i275-82. (PMID: 21685081) Genes Dev. 1993 Jan;7(1):42-54. (PMID: 8422987) Genetics. 2013 Sep;195(1):289-91. (PMID: 23833182) RSC Chem Biol. 2021 Jul 2;2(5):1370-1383. (PMID: 34704043) Dev Biol. 1993 Jan;155(1):235-49. (PMID: 8416836) Proc Natl Acad Sci U S A. 2003 Mar 4;100(5):2556-61. (PMID: 12589026) Nat Protoc. 2006;1(6):2742-5. (PMID: 17406530) Development. 1999 Dec;126(24):5645-57. (PMID: 10572041) Elife. 2017 Feb 17;6:. (PMID: 28211790) Nature. 2006 Sep 21;443(7109):359-63. (PMID: 16964239) Genetics. 1965 Apr;51:659-78. (PMID: 14330702) Genes Dev. 2016 Mar 1;30(5):594-609. (PMID: 26944682) Cell. 2014 Feb 13;156(4):812-24. (PMID: 24529382) RNA. 2006 May;12(5):797-806. (PMID: 16556935) Cell Rep. 2013 Jul 11;4(1):220-8. (PMID: 23827738) Nature. 1985 Feb 21-27;313(6004):703-6. (PMID: 2579337) Genetics. 2018 Mar;208(3):853-874. (PMID: 29487145) Development. 2000 Feb;127(4):881-92. (PMID: 10648246) Cold Spring Harb Perspect Biol. 2012 Oct 01;4(10):. (PMID: 22865893) Methods Mol Biol. 2015;1328:1-20. (PMID: 26324426) RNA. 2012 May;18(5):871-85. (PMID: 22454536) Nature. 2007 Nov 8;450(7167):203-18. (PMID: 17994087) Methods Mol Biol. 2021;2167:287-301. (PMID: 32712926) Nucleic Acids Res. 2007 Jul;35(Web Server issue):W345-9. (PMID: 17631615) RNA. 2018 Jan;24(1):98-113. (PMID: 29079635) Genome Biol. 2015 Jan 29;16:20. (PMID: 25630241) Trends Biotechnol. 2019 Jul;37(7):761-774. (PMID: 30654913) Genome Res. 2005 Jan;15(1):1-18. (PMID: 15632085) Nature. 2004 Sep 23;431(7007):471-6. (PMID: 15386015) Genes Dev. 1987 Nov;1(9):913-23. (PMID: 3428601) Methods Mol Biol. 2017;1642:195-209. (PMID: 28815502) Proc Natl Acad Sci U S A. 2007 Sep 4;104(36):14283-8. (PMID: 17709748) BMC Genomics. 2006 Jul 12;7:176. (PMID: 16836751) Cell. 1991 Oct 4;67(1):59-77. (PMID: 1913820) Nature. 2002 Jul 11;418(6894):236-43. (PMID: 12110900) RNA. 2017 Oct;23(10):1582-1591. (PMID: 28698239) Nature. 2011 Mar 24;471(7339):473-9. (PMID: 21179090) Cell Biosci. 2014 Oct 14;4(1):63. (PMID: 25364499) Genetics. 2015 Nov;201(3):843-52. (PMID: 26320097) Mol Cell. 1999 Jul;4(1):117-22. (PMID: 10445033) Nat Cell Biol. 2006 Aug;8(8):793-802. (PMID: 16862146) RNA Biol. 2018 Feb 1;15(2):231-241. (PMID: 29106331) Mol Biosyst. 2011 Aug;7(8):2419-27. (PMID: 21603688) Nucleic Acids Res. 2016 Jan 4;44(D1):D717-25. (PMID: 26590259) Genes Dev. 2006 May 1;20(9):1050-6. (PMID: 16651651) Nature. 2014 Aug 28;512(7515):393-9. (PMID: 24670639) Dev Dyn. 2008 Oct;237(10):3040-50. (PMID: 18816856) Genetics. 1980 Feb;94(2):383-423. (PMID: 6771185) Cell. 2009 Feb 20;136(4):763-76. (PMID: 19239894) Nucleic Acids Res. 2013 Apr 1;41(6):e74. (PMID: 23335781) EMBO J. 1991 Aug;10(8):2259-66. (PMID: 1712294) Nat Methods. 2009 May;6(5):343-5. (PMID: 19363495) Elife. 2014 Dec 15;3:e04766. (PMID: 25497837) Nat Biotechnol. 2020 Feb;38(2):169-175. (PMID: 31873216) Elife. 2020 Jun 22;9:. (PMID: 32568073) Cell. 1989 Mar 24;56(6):997-1010. (PMID: 2493994) Genesis. 2002 Sep-Oct;34(1-2):1-15. (PMID: 12324939) Nat Commun. 2019 Jan 31;10(1):521. (PMID: 30705266) Nat Biotechnol. 2000 Aug;18(8):896-8. (PMID: 10932163) Cell. 1994 Jul 29;78(2):291-301. (PMID: 8044841) |
| معلومات مُعتمدة: | Department of Molecular Biosciences, and the Rice Foundation; NU Office for Research; P30 CA060553 United States CA NCI NIH HHS; R35GM118144 United States NH NIH HHS; F32 GM122349 United States GM NIGMS NIH HHS; 597491 Simons Foundation; Chemistry for Life Processes Institute; 1764421 National Science Foundation; RRID:SCR_017767 Northwestern's Biological Imaging Facility; CA060553 NCI Cancer Center Support Grant |
| فهرسة مساهمة: | Keywords: gene expression; knockdown; noncoding RNAs; ribozyme; splice variants |
| المشرفين على المادة: | 0 (RNA, Catalytic) 0 (hammerhead ribozyme) |
| تواريخ الأحداث: | Date Created: 20240503 Date Completed: 20240807 Latest Revision: 20240809 |
| رمز التحديث: | 20240809 |
| مُعرف محوري في PubMed: | PMC11304983 |
| DOI: | 10.1093/genetics/iyae067 |
| PMID: | 38701221 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 1943-2631 |
|---|---|
| DOI: | 10.1093/genetics/iyae067 |