Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage

The first step in CRISPR-Cas9-mediated genome editing is the cleavage of target DNA sequences that are complementary to so-called spacer sequences in CRISPR guide RNAs (gRNAs). However, some DNA sequences are refractory to CRISPR-Cas9 cleavage, which is at least in part due to gRNA misfolding. To ov...

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Autores principales: Riesenberg, Stephan, Helmbrecht, Nelly, Kanis, Philipp, Maricic, Tomislav, Pääbo, Svante
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
Materias:
Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8789806/
https://www.ncbi.nlm.nih.gov/pubmed/35078986
http://dx.doi.org/10.1038/s41467-022-28137-7
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author Riesenberg, Stephan
Helmbrecht, Nelly
Kanis, Philipp
Maricic, Tomislav
Pääbo, Svante
author_facet Riesenberg, Stephan
Helmbrecht, Nelly
Kanis, Philipp
Maricic, Tomislav
Pääbo, Svante
author_sort Riesenberg, Stephan
collection PubMed
description The first step in CRISPR-Cas9-mediated genome editing is the cleavage of target DNA sequences that are complementary to so-called spacer sequences in CRISPR guide RNAs (gRNAs). However, some DNA sequences are refractory to CRISPR-Cas9 cleavage, which is at least in part due to gRNA misfolding. To overcome this problem, we have engineered gRNAs with highly stable hairpins in their constant parts and further enhanced their stability by chemical modifications. The ‘Genome-editing Optimized Locked Design’ (GOLD)-gRNA increases genome editing efficiency up to around 1000-fold (from 0.08 to 80.5%) with a mean increase across different other targets of 7.4-fold. We anticipate that this improved gRNA will allow efficient editing regardless of spacer sequence composition and will be especially useful if a desired genomic site is difficult to edit.
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spelling pubmed-87898062022-02-07 Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage Riesenberg, Stephan Helmbrecht, Nelly Kanis, Philipp Maricic, Tomislav Pääbo, Svante Nat Commun Article The first step in CRISPR-Cas9-mediated genome editing is the cleavage of target DNA sequences that are complementary to so-called spacer sequences in CRISPR guide RNAs (gRNAs). However, some DNA sequences are refractory to CRISPR-Cas9 cleavage, which is at least in part due to gRNA misfolding. To overcome this problem, we have engineered gRNAs with highly stable hairpins in their constant parts and further enhanced their stability by chemical modifications. The ‘Genome-editing Optimized Locked Design’ (GOLD)-gRNA increases genome editing efficiency up to around 1000-fold (from 0.08 to 80.5%) with a mean increase across different other targets of 7.4-fold. We anticipate that this improved gRNA will allow efficient editing regardless of spacer sequence composition and will be especially useful if a desired genomic site is difficult to edit. Nature Publishing Group UK 2022-01-25 /pmc/articles/PMC8789806/ /pubmed/35078986 http://dx.doi.org/10.1038/s41467-022-28137-7 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open Access This 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Riesenberg, Stephan
Helmbrecht, Nelly
Kanis, Philipp
Maricic, Tomislav
Pääbo, Svante
Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage
title Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage
title_full Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage
title_fullStr Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage
title_full_unstemmed Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage
title_short Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage
title_sort improved grna secondary structures allow editing of target sites resistant to crispr-cas9 cleavage
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8789806/
https://www.ncbi.nlm.nih.gov/pubmed/35078986
http://dx.doi.org/10.1038/s41467-022-28137-7
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