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Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity, but at the cost of the on-target activity. DNA unwinding is a checkpoint before cleav...

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Autores principales: Okafor, Ikenna C, Singh, Digvijay, Wang, Yanbo, Jung, Minhee, Wang, Haobo, Mallon, John, Bailey, Scott, Lee, Jungjoon K, Ha, Taekjip
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145707/
https://www.ncbi.nlm.nih.gov/pubmed/31713616
http://dx.doi.org/10.1093/nar/gkz1058
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author Okafor, Ikenna C
Singh, Digvijay
Wang, Yanbo
Jung, Minhee
Wang, Haobo
Mallon, John
Bailey, Scott
Lee, Jungjoon K
Ha, Taekjip
author_facet Okafor, Ikenna C
Singh, Digvijay
Wang, Yanbo
Jung, Minhee
Wang, Haobo
Mallon, John
Bailey, Scott
Lee, Jungjoon K
Ha, Taekjip
author_sort Okafor, Ikenna C
collection PubMed
description Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity, but at the cost of the on-target activity. DNA unwinding is a checkpoint before cleavage by Cas9, and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in engineered Cas9s. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used, but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two non-matching guanines added to the 5′ end were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5′ end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.
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spelling pubmed-71457072020-04-13 Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding Okafor, Ikenna C Singh, Digvijay Wang, Yanbo Jung, Minhee Wang, Haobo Mallon, John Bailey, Scott Lee, Jungjoon K Ha, Taekjip Nucleic Acids Res RNA and RNA-Protein Complexes Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity, but at the cost of the on-target activity. DNA unwinding is a checkpoint before cleavage by Cas9, and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in engineered Cas9s. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used, but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two non-matching guanines added to the 5′ end were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5′ end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering. Oxford University Press 2019-12-16 2019-11-12 /pmc/articles/PMC7145707/ /pubmed/31713616 http://dx.doi.org/10.1093/nar/gkz1058 Text en © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle RNA and RNA-Protein Complexes
Okafor, Ikenna C
Singh, Digvijay
Wang, Yanbo
Jung, Minhee
Wang, Haobo
Mallon, John
Bailey, Scott
Lee, Jungjoon K
Ha, Taekjip
Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding
title Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding
title_full Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding
title_fullStr Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding
title_full_unstemmed Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding
title_short Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding
title_sort single molecule analysis of effects of non-canonical guide rnas and specificity-enhancing mutations on cas9-induced dna unwinding
topic RNA and RNA-Protein Complexes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145707/
https://www.ncbi.nlm.nih.gov/pubmed/31713616
http://dx.doi.org/10.1093/nar/gkz1058
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