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Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing

A major challenge confronting the clinical application of site-directed RNA editing (SDRE) is the design of small guide RNAs (gRNAs) that can drive efficient editing. Although many gRNA designs have effectively recruited endogenous Adenosine Deaminases that Act on RNA (ADARs), most of them exceed th...

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Autores principales: Diaz Quiroz, Juan Felipe, Ojha, Namrata, Shayhidin, Elnur E, De Silva, Dasuni, Dabney, Jesse, Lancaster, Amy, Coull, James, Milstein, Stuart, Fraley, Andrew W, Brown, Christopher R, Rosenthal, Joshua J C
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10123091/
https://www.ncbi.nlm.nih.gov/pubmed/36840708
http://dx.doi.org/10.1093/nar/gkad098
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author Diaz Quiroz, Juan Felipe
Ojha, Namrata
Shayhidin, Elnur E
De Silva, Dasuni
Dabney, Jesse
Lancaster, Amy
Coull, James
Milstein, Stuart
Fraley, Andrew W
Brown, Christopher R
Rosenthal, Joshua J C
author_facet Diaz Quiroz, Juan Felipe
Ojha, Namrata
Shayhidin, Elnur E
De Silva, Dasuni
Dabney, Jesse
Lancaster, Amy
Coull, James
Milstein, Stuart
Fraley, Andrew W
Brown, Christopher R
Rosenthal, Joshua J C
author_sort Diaz Quiroz, Juan Felipe
collection PubMed
description A major challenge confronting the clinical application of site-directed RNA editing (SDRE) is the design of small guide RNAs (gRNAs) that can drive efficient editing. Although many gRNA designs have effectively recruited endogenous Adenosine Deaminases that Act on RNA (ADARs), most of them exceed the size of currently FDA-approved antisense oligos. We developed an unbiased in vitro selection assay to identify short gRNAs that promote superior RNA editing of a premature termination codon. The selection assay relies on hairpin substrates in which the target sequence is linked to partially randomized gRNAs in the same molecule, so that gRNA sequences that promote editing can be identified by sequencing. These RNA substrates were incubated in vitro with ADAR2 and the edited products were selected using amplification refractory mutation system PCR and used to regenerate the substrates for a new round of selection. After nine repetitions, hairpins which drove superior editing were identified. When gRNAs of these hairpins were delivered in trans, eight of the top ten short gRNAs drove superior editing both in vitro and in cellula. These results show that efficient small gRNAs can be selected using our approach, an important advancement for the clinical application of SDRE.
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spelling pubmed-101230912023-04-25 Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing Diaz Quiroz, Juan Felipe Ojha, Namrata Shayhidin, Elnur E De Silva, Dasuni Dabney, Jesse Lancaster, Amy Coull, James Milstein, Stuart Fraley, Andrew W Brown, Christopher R Rosenthal, Joshua J C Nucleic Acids Res Methods Online A major challenge confronting the clinical application of site-directed RNA editing (SDRE) is the design of small guide RNAs (gRNAs) that can drive efficient editing. Although many gRNA designs have effectively recruited endogenous Adenosine Deaminases that Act on RNA (ADARs), most of them exceed the size of currently FDA-approved antisense oligos. We developed an unbiased in vitro selection assay to identify short gRNAs that promote superior RNA editing of a premature termination codon. The selection assay relies on hairpin substrates in which the target sequence is linked to partially randomized gRNAs in the same molecule, so that gRNA sequences that promote editing can be identified by sequencing. These RNA substrates were incubated in vitro with ADAR2 and the edited products were selected using amplification refractory mutation system PCR and used to regenerate the substrates for a new round of selection. After nine repetitions, hairpins which drove superior editing were identified. When gRNAs of these hairpins were delivered in trans, eight of the top ten short gRNAs drove superior editing both in vitro and in cellula. These results show that efficient small gRNAs can be selected using our approach, an important advancement for the clinical application of SDRE. Oxford University Press 2023-02-25 /pmc/articles/PMC10123091/ /pubmed/36840708 http://dx.doi.org/10.1093/nar/gkad098 Text en © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Diaz Quiroz, Juan Felipe
Ojha, Namrata
Shayhidin, Elnur E
De Silva, Dasuni
Dabney, Jesse
Lancaster, Amy
Coull, James
Milstein, Stuart
Fraley, Andrew W
Brown, Christopher R
Rosenthal, Joshua J C
Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing
title Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing
title_full Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing
title_fullStr Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing
title_full_unstemmed Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing
title_short Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing
title_sort development of a selection assay for small guide rnas that drive efficient site-directed rna editing
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10123091/
https://www.ncbi.nlm.nih.gov/pubmed/36840708
http://dx.doi.org/10.1093/nar/gkad098
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