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Sidsel Rugberg Alsing

Efficient Knockdown and Lack of Passenger Strand Activity by Dicer-Independent shRNAs Expressed from Pol II-Driven MicroRNA Scaffolds

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Efficient Knockdown and Lack of Passenger Strand Activity by Dicer-Independent shRNAs Expressed from Pol II-Driven MicroRNA Scaffolds. / Kaadt, Erik; Alsing, Sidsel; Cecchi, Claudia R.; Damgaard, Christian Kroun; Corydon, Thomas J.; Aagaard, Lars.

I: Molecular Therapy - Nucleic Acids, Bind 14, 01.03.2019, s. 318-328.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

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@article{e4ae4aa84d5a46b2a3cf5de53b7b176c,
title = "Efficient Knockdown and Lack of Passenger Strand Activity by Dicer-Independent shRNAs Expressed from Pol II-Driven MicroRNA Scaffolds",
abstract = "The expression of short hairpin RNAs (shRNAs) may result in unwanted activity from the co-processed passenger strand. Recent studies have shown that shortening the stem of conventional shRNAs abolishes passenger strand release. These Dicer-independent shRNAs, expressed from RNA polymerase III (Pol III) promoters, rely on Ago2 processing in resemblance to miR-451. Using strand-specific reporters, we tested two designs, and our results support the loss of passenger strand activity. We demonstrate that artificial primary microRNA (pri-miRNA) transcripts, expressed from Pol II promoters, can potently silence a gene of choice. Among six different scaffolds tested, miR-324 and miR-451 were readily re-targeted to direct efficient knockdown from either a CMV or a U1 snRNA promoter. Importantly, the miR-shRNAs have no passenger strand activity and remain active in Dicer-knockout cells. Our vectors are straightforward to design, as we replace the pre-miR-324 or -451 sequences with a Dicer-independent shRNA mimicking miR-451 with unpaired A-C nucleotides at the base. The use of Pol II promoters allows for controlled expression, while the inclusion of pri-miRNA sequences likely requires Drosha processing and, as such, mimics microRNA biogenesis. Since this improved and tunable system bypasses the requirement for Dicer activity and abolishes passenger strand activity completely, it will likely prove favorable in both research and therapeutic applications in terms of versatility and enhanced safety.",
keywords = "agoshRNA, agshRNA, Dicer-independent shRNA, Drosha, miR-324, miR-451, passenger strand activity, Pol-II driven miRNA scaffold, RNAi, U1 promoter",
author = "Erik Kaadt and Sidsel Alsing and Cecchi, {Claudia R.} and Damgaard, {Christian Kroun} and Corydon, {Thomas J.} and Lars Aagaard",
year = "2019",
month = "3",
day = "1",
doi = "10.1016/j.omtn.2018.11.013",
language = "English",
volume = "14",
pages = "318--328",
journal = "Molecular Therapy - Nucleic Acids",
issn = "2162-2531",
publisher = "Nature Publishing Group",

}

RIS

TY - JOUR

T1 - Efficient Knockdown and Lack of Passenger Strand Activity by Dicer-Independent shRNAs Expressed from Pol II-Driven MicroRNA Scaffolds

AU - Kaadt, Erik

AU - Alsing, Sidsel

AU - Cecchi, Claudia R.

AU - Damgaard, Christian Kroun

AU - Corydon, Thomas J.

AU - Aagaard, Lars

PY - 2019/3/1

Y1 - 2019/3/1

N2 - The expression of short hairpin RNAs (shRNAs) may result in unwanted activity from the co-processed passenger strand. Recent studies have shown that shortening the stem of conventional shRNAs abolishes passenger strand release. These Dicer-independent shRNAs, expressed from RNA polymerase III (Pol III) promoters, rely on Ago2 processing in resemblance to miR-451. Using strand-specific reporters, we tested two designs, and our results support the loss of passenger strand activity. We demonstrate that artificial primary microRNA (pri-miRNA) transcripts, expressed from Pol II promoters, can potently silence a gene of choice. Among six different scaffolds tested, miR-324 and miR-451 were readily re-targeted to direct efficient knockdown from either a CMV or a U1 snRNA promoter. Importantly, the miR-shRNAs have no passenger strand activity and remain active in Dicer-knockout cells. Our vectors are straightforward to design, as we replace the pre-miR-324 or -451 sequences with a Dicer-independent shRNA mimicking miR-451 with unpaired A-C nucleotides at the base. The use of Pol II promoters allows for controlled expression, while the inclusion of pri-miRNA sequences likely requires Drosha processing and, as such, mimics microRNA biogenesis. Since this improved and tunable system bypasses the requirement for Dicer activity and abolishes passenger strand activity completely, it will likely prove favorable in both research and therapeutic applications in terms of versatility and enhanced safety.

AB - The expression of short hairpin RNAs (shRNAs) may result in unwanted activity from the co-processed passenger strand. Recent studies have shown that shortening the stem of conventional shRNAs abolishes passenger strand release. These Dicer-independent shRNAs, expressed from RNA polymerase III (Pol III) promoters, rely on Ago2 processing in resemblance to miR-451. Using strand-specific reporters, we tested two designs, and our results support the loss of passenger strand activity. We demonstrate that artificial primary microRNA (pri-miRNA) transcripts, expressed from Pol II promoters, can potently silence a gene of choice. Among six different scaffolds tested, miR-324 and miR-451 were readily re-targeted to direct efficient knockdown from either a CMV or a U1 snRNA promoter. Importantly, the miR-shRNAs have no passenger strand activity and remain active in Dicer-knockout cells. Our vectors are straightforward to design, as we replace the pre-miR-324 or -451 sequences with a Dicer-independent shRNA mimicking miR-451 with unpaired A-C nucleotides at the base. The use of Pol II promoters allows for controlled expression, while the inclusion of pri-miRNA sequences likely requires Drosha processing and, as such, mimics microRNA biogenesis. Since this improved and tunable system bypasses the requirement for Dicer activity and abolishes passenger strand activity completely, it will likely prove favorable in both research and therapeutic applications in terms of versatility and enhanced safety.

KW - agoshRNA

KW - agshRNA

KW - Dicer-independent shRNA

KW - Drosha

KW - miR-324

KW - miR-451

KW - passenger strand activity

KW - Pol-II driven miRNA scaffold

KW - RNAi

KW - U1 promoter

UR - http://www.scopus.com/inward/record.url?scp=85059868564&partnerID=8YFLogxK

U2 - 10.1016/j.omtn.2018.11.013

DO - 10.1016/j.omtn.2018.11.013

M3 - Journal article

C2 - 30654192

AN - SCOPUS:85059868564

VL - 14

SP - 318

EP - 328

JO - Molecular Therapy - Nucleic Acids

JF - Molecular Therapy - Nucleic Acids

SN - 2162-2531

ER -