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CRISPR guides induce gene silencing in plants in the absence of Cas
BACKGROUND: RNA-targeting CRISPR-Cas can provide potential advantages over DNA editing, such as avoiding pleiotropic effects of genome editing, providing precise spatiotemporal regulation, and expanded function including antiviral immunity. RESULTS: Here, we report the use of CRISPR-Cas13 in plants...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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BioMed Central
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8722000/ https://www.ncbi.nlm.nih.gov/pubmed/34980227 http://dx.doi.org/10.1186/s13059-021-02586-7 |
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author | Sharma, Veerendra Kumar Marla, Sandeep Zheng, Wenguang Mishra, Divya Huang, Jun Zhang, Wei Morris, Geoffrey Preston Cook, David Edward |
author_facet | Sharma, Veerendra Kumar Marla, Sandeep Zheng, Wenguang Mishra, Divya Huang, Jun Zhang, Wei Morris, Geoffrey Preston Cook, David Edward |
author_sort | Sharma, Veerendra Kumar |
collection | PubMed |
description | BACKGROUND: RNA-targeting CRISPR-Cas can provide potential advantages over DNA editing, such as avoiding pleiotropic effects of genome editing, providing precise spatiotemporal regulation, and expanded function including antiviral immunity. RESULTS: Here, we report the use of CRISPR-Cas13 in plants to reduce both viral and endogenous RNA. Unexpectedly, we observe that crRNA designed to guide Cas13 could, in the absence of the Cas13 protein, cause substantial reduction in RNA levels as well. We demonstrate Cas13-independent guide-induced gene silencing (GIGS) in three plant species, including stable transgenic Arabidopsis. Small RNA sequencing during GIGS identifies the production of small RNA that extend beyond the crRNA expressed sequence in samples expressing multi-guide crRNA. Additionally, we demonstrate that mismatches in guide sequences at position 10 and 11 abolish GIGS. Finally, we show that GIGS is elicited by guides that lack the Cas13 direct repeat and can extend to Cas9 designed crRNA of at least 28 base pairs, indicating that GIGS can be elicited through a variety of guide designs and is not dependent on Cas13 crRNA sequences or design. CONCLUSIONS: Collectively, our results suggest that GIGS utilizes endogenous RNAi machinery despite the fact that crRNA are unlike canonical triggers of RNAi such as miRNA, hairpins, or long double-stranded RNA. Given similar evidence of Cas13-independent silencing in an insect system, it is likely GIGS is active across many eukaryotes. Our results show that GIGS offers a novel and flexible approach to RNA reduction with potential benefits over existing technologies for crop improvement and functional genomics. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13059-021-02586-7. |
format | Online Article Text |
id | pubmed-8722000 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-87220002022-01-06 CRISPR guides induce gene silencing in plants in the absence of Cas Sharma, Veerendra Kumar Marla, Sandeep Zheng, Wenguang Mishra, Divya Huang, Jun Zhang, Wei Morris, Geoffrey Preston Cook, David Edward Genome Biol Research BACKGROUND: RNA-targeting CRISPR-Cas can provide potential advantages over DNA editing, such as avoiding pleiotropic effects of genome editing, providing precise spatiotemporal regulation, and expanded function including antiviral immunity. RESULTS: Here, we report the use of CRISPR-Cas13 in plants to reduce both viral and endogenous RNA. Unexpectedly, we observe that crRNA designed to guide Cas13 could, in the absence of the Cas13 protein, cause substantial reduction in RNA levels as well. We demonstrate Cas13-independent guide-induced gene silencing (GIGS) in three plant species, including stable transgenic Arabidopsis. Small RNA sequencing during GIGS identifies the production of small RNA that extend beyond the crRNA expressed sequence in samples expressing multi-guide crRNA. Additionally, we demonstrate that mismatches in guide sequences at position 10 and 11 abolish GIGS. Finally, we show that GIGS is elicited by guides that lack the Cas13 direct repeat and can extend to Cas9 designed crRNA of at least 28 base pairs, indicating that GIGS can be elicited through a variety of guide designs and is not dependent on Cas13 crRNA sequences or design. CONCLUSIONS: Collectively, our results suggest that GIGS utilizes endogenous RNAi machinery despite the fact that crRNA are unlike canonical triggers of RNAi such as miRNA, hairpins, or long double-stranded RNA. Given similar evidence of Cas13-independent silencing in an insect system, it is likely GIGS is active across many eukaryotes. Our results show that GIGS offers a novel and flexible approach to RNA reduction with potential benefits over existing technologies for crop improvement and functional genomics. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13059-021-02586-7. BioMed Central 2022-01-03 /pmc/articles/PMC8722000/ /pubmed/34980227 http://dx.doi.org/10.1186/s13059-021-02586-7 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Sharma, Veerendra Kumar Marla, Sandeep Zheng, Wenguang Mishra, Divya Huang, Jun Zhang, Wei Morris, Geoffrey Preston Cook, David Edward CRISPR guides induce gene silencing in plants in the absence of Cas |
title | CRISPR guides induce gene silencing in plants in the absence of Cas |
title_full | CRISPR guides induce gene silencing in plants in the absence of Cas |
title_fullStr | CRISPR guides induce gene silencing in plants in the absence of Cas |
title_full_unstemmed | CRISPR guides induce gene silencing in plants in the absence of Cas |
title_short | CRISPR guides induce gene silencing in plants in the absence of Cas |
title_sort | crispr guides induce gene silencing in plants in the absence of cas |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8722000/ https://www.ncbi.nlm.nih.gov/pubmed/34980227 http://dx.doi.org/10.1186/s13059-021-02586-7 |
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