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Improving Precise Genome Editing Using Donor DNA/gRNA Hybrid Duplex Generated by Complementary Bases

In precise genome editing, site-specific DNA double-strand breaks (DSBs) induced by the CRISPR/Cas9 system are repaired via homology-directed repair (HDR) using exogenous donor DNA templates. However, the low efficiency of HDR-mediated genome editing is a barrier to widespread use. In this study, we...

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Detalles Bibliográficos
Autores principales: Aiba, Wataru, Amai, Takamitsu, Ueda, Mitsuyoshi, Kuroda, Kouichi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9687273/
https://www.ncbi.nlm.nih.gov/pubmed/36358971
http://dx.doi.org/10.3390/biom12111621
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author Aiba, Wataru
Amai, Takamitsu
Ueda, Mitsuyoshi
Kuroda, Kouichi
author_facet Aiba, Wataru
Amai, Takamitsu
Ueda, Mitsuyoshi
Kuroda, Kouichi
author_sort Aiba, Wataru
collection PubMed
description In precise genome editing, site-specific DNA double-strand breaks (DSBs) induced by the CRISPR/Cas9 system are repaired via homology-directed repair (HDR) using exogenous donor DNA templates. However, the low efficiency of HDR-mediated genome editing is a barrier to widespread use. In this study, we created a donor DNA/guide RNA (gRNA) hybrid duplex (DGybrid) that was composed of sequence-extended gRNA and single-stranded oligodeoxynucleotide (ssODN) combined with complementary bases without chemical modifications to increase the concentration of donor DNA at the cleavage site. The efficiency of genome editing using DGybrid was evaluated in Saccharomyces cerevisiae. The results show a 1.8-fold (from 35% to 62%) improvement in HDR-mediated editing efficiency compared to genome editing in which gRNA and donor DNA were introduced separately. In addition, analysis of the nucleic acid introduction efficiency using flow cytometry indicated that both RNA and ssODNs are efficiently incorporated into cells together by using the DNA/RNA hybrid. Our technique would be preferred as a universal and concise tool for improving the efficiency of HDR-mediated genome editing.
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spelling pubmed-96872732022-11-25 Improving Precise Genome Editing Using Donor DNA/gRNA Hybrid Duplex Generated by Complementary Bases Aiba, Wataru Amai, Takamitsu Ueda, Mitsuyoshi Kuroda, Kouichi Biomolecules Article In precise genome editing, site-specific DNA double-strand breaks (DSBs) induced by the CRISPR/Cas9 system are repaired via homology-directed repair (HDR) using exogenous donor DNA templates. However, the low efficiency of HDR-mediated genome editing is a barrier to widespread use. In this study, we created a donor DNA/guide RNA (gRNA) hybrid duplex (DGybrid) that was composed of sequence-extended gRNA and single-stranded oligodeoxynucleotide (ssODN) combined with complementary bases without chemical modifications to increase the concentration of donor DNA at the cleavage site. The efficiency of genome editing using DGybrid was evaluated in Saccharomyces cerevisiae. The results show a 1.8-fold (from 35% to 62%) improvement in HDR-mediated editing efficiency compared to genome editing in which gRNA and donor DNA were introduced separately. In addition, analysis of the nucleic acid introduction efficiency using flow cytometry indicated that both RNA and ssODNs are efficiently incorporated into cells together by using the DNA/RNA hybrid. Our technique would be preferred as a universal and concise tool for improving the efficiency of HDR-mediated genome editing. MDPI 2022-11-03 /pmc/articles/PMC9687273/ /pubmed/36358971 http://dx.doi.org/10.3390/biom12111621 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Aiba, Wataru
Amai, Takamitsu
Ueda, Mitsuyoshi
Kuroda, Kouichi
Improving Precise Genome Editing Using Donor DNA/gRNA Hybrid Duplex Generated by Complementary Bases
title Improving Precise Genome Editing Using Donor DNA/gRNA Hybrid Duplex Generated by Complementary Bases
title_full Improving Precise Genome Editing Using Donor DNA/gRNA Hybrid Duplex Generated by Complementary Bases
title_fullStr Improving Precise Genome Editing Using Donor DNA/gRNA Hybrid Duplex Generated by Complementary Bases
title_full_unstemmed Improving Precise Genome Editing Using Donor DNA/gRNA Hybrid Duplex Generated by Complementary Bases
title_short Improving Precise Genome Editing Using Donor DNA/gRNA Hybrid Duplex Generated by Complementary Bases
title_sort improving precise genome editing using donor dna/grna hybrid duplex generated by complementary bases
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9687273/
https://www.ncbi.nlm.nih.gov/pubmed/36358971
http://dx.doi.org/10.3390/biom12111621
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