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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...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2023
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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. |
format | Online Article Text |
id | pubmed-10123091 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
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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