Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases

High-fidelity SpCas9 variants (eSpCas9 and SpCas9-HF1) have been engineered to reduce off-target effects. We found that changes in guide RNA length induced significant reductions in the editing activities of SpCas9 variants in plant cells. Single guide RNAs harboring precise, perfectly matched 20-nu...

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Autores principales: Zhang, Dingbo, Zhang, Huawei, Li, Tingdong, Chen, Kunling, Qiu, Jin-Long, Gao, Caixia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Method
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5637269/
https://www.ncbi.nlm.nih.gov/pubmed/29020979
http://dx.doi.org/10.1186/s13059-017-1325-9
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author Zhang, Dingbo
Zhang, Huawei
Li, Tingdong
Chen, Kunling
Qiu, Jin-Long
Gao, Caixia
author_facet Zhang, Dingbo
Zhang, Huawei
Li, Tingdong
Chen, Kunling
Qiu, Jin-Long
Gao, Caixia
author_sort Zhang, Dingbo
collection PubMed
description High-fidelity SpCas9 variants (eSpCas9 and SpCas9-HF1) have been engineered to reduce off-target effects. We found that changes in guide RNA length induced significant reductions in the editing activities of SpCas9 variants in plant cells. Single guide RNAs harboring precise, perfectly matched 20-nucleotide guide sequences are necessary for high on-target editing activities of eSpCas9 and SpCas9-HF1. Precise 20-nucleotide guide sequences derived from tRNA–sgRNA precursors enable robust on-target editing by these variants with enhanced specificity. Our work reveals an effective way of enhancing the use of the high-fidelity SpCas9 nucleases for efficient and precise genome engineering. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13059-017-1325-9) contains supplementary material, which is available to authorized users.
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spelling pubmed-56372692017-10-18 Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases Zhang, Dingbo Zhang, Huawei Li, Tingdong Chen, Kunling Qiu, Jin-Long Gao, Caixia Genome Biol Method High-fidelity SpCas9 variants (eSpCas9 and SpCas9-HF1) have been engineered to reduce off-target effects. We found that changes in guide RNA length induced significant reductions in the editing activities of SpCas9 variants in plant cells. Single guide RNAs harboring precise, perfectly matched 20-nucleotide guide sequences are necessary for high on-target editing activities of eSpCas9 and SpCas9-HF1. Precise 20-nucleotide guide sequences derived from tRNA–sgRNA precursors enable robust on-target editing by these variants with enhanced specificity. Our work reveals an effective way of enhancing the use of the high-fidelity SpCas9 nucleases for efficient and precise genome engineering. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13059-017-1325-9) contains supplementary material, which is available to authorized users. BioMed Central 2017-10-11 /pmc/articles/PMC5637269/ /pubmed/29020979 http://dx.doi.org/10.1186/s13059-017-1325-9 Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Method
Zhang, Dingbo
Zhang, Huawei
Li, Tingdong
Chen, Kunling
Qiu, Jin-Long
Gao, Caixia
Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases
title Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases
title_full Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases
title_fullStr Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases
title_full_unstemmed Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases
title_short Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases
title_sort perfectly matched 20-nucleotide guide rna sequences enable robust genome editing using high-fidelity spcas9 nucleases
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5637269/
https://www.ncbi.nlm.nih.gov/pubmed/29020979
http://dx.doi.org/10.1186/s13059-017-1325-9
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