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Mechanisms of precise genome editing using oligonucleotide donors
The use of programmable meganucleases is transforming genome editing and functional genomics. CRISPR/Cas9 was developed such that targeted genomic lesions could be introduced in vivo with unprecedented ease. In the presence of homology donors, these lesions facilitate high-efficiency precise genome...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory Press
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495063/ https://www.ncbi.nlm.nih.gov/pubmed/28356322 http://dx.doi.org/10.1101/gr.214775.116 |
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author | Kan, Yinan Ruis, Brian Takasugi, Taylor Hendrickson, Eric A. |
author_facet | Kan, Yinan Ruis, Brian Takasugi, Taylor Hendrickson, Eric A. |
author_sort | Kan, Yinan |
collection | PubMed |
description | The use of programmable meganucleases is transforming genome editing and functional genomics. CRISPR/Cas9 was developed such that targeted genomic lesions could be introduced in vivo with unprecedented ease. In the presence of homology donors, these lesions facilitate high-efficiency precise genome editing (PGE) via homology-directed repair (HDR) pathways. However, the identity and hierarchy of the HDR (sub)pathways leading to the formation of PGE products remain elusive. Here, we established a green to blue fluorescent protein conversion system to systematically characterize oligodeoxynucleotide (ODN)-mediated PGE using Cas9 and its nickase variants in human cells. We demonstrate that, unlike double-stranded DNA (dsDNA) donors with central heterologies, ODNs generated short conversion tracts with Gaussian-like distributions. Interestingly, single-nick–induced PGE using ODN donors produced conversion tracts biased either mostly uni- or bidirectional depending on the relative strandedness of the ODNs and the nick. Moreover, the ODNs were physically incorporated into the genome only in the bidirectional, but not in the unidirectional, conversion pathway. In the presence of double-stranded genomic lesions, the unidirectional conversion pathway was preferentially utilized even though the knock-in mutation could theoretically have been converted by both pathways. Collectively, our results suggest that ODN-mediated PGE utilizes synthesis-dependent strand annealing and single-stranded DNA incorporation pathways. Both of these pathways generate short conversion tracts with Gaussian-like distributions. Although synthesis-dependent strand annealing is preferentially utilized, our work unequivocally establishes the existence of a single-stranded DNA incorporation pathway in human cells. This work extends the paradigms of HDR-mediated gene conversion and establishes guidelines for PGE in human cells. |
format | Online Article Text |
id | pubmed-5495063 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-54950632018-01-01 Mechanisms of precise genome editing using oligonucleotide donors Kan, Yinan Ruis, Brian Takasugi, Taylor Hendrickson, Eric A. Genome Res Research The use of programmable meganucleases is transforming genome editing and functional genomics. CRISPR/Cas9 was developed such that targeted genomic lesions could be introduced in vivo with unprecedented ease. In the presence of homology donors, these lesions facilitate high-efficiency precise genome editing (PGE) via homology-directed repair (HDR) pathways. However, the identity and hierarchy of the HDR (sub)pathways leading to the formation of PGE products remain elusive. Here, we established a green to blue fluorescent protein conversion system to systematically characterize oligodeoxynucleotide (ODN)-mediated PGE using Cas9 and its nickase variants in human cells. We demonstrate that, unlike double-stranded DNA (dsDNA) donors with central heterologies, ODNs generated short conversion tracts with Gaussian-like distributions. Interestingly, single-nick–induced PGE using ODN donors produced conversion tracts biased either mostly uni- or bidirectional depending on the relative strandedness of the ODNs and the nick. Moreover, the ODNs were physically incorporated into the genome only in the bidirectional, but not in the unidirectional, conversion pathway. In the presence of double-stranded genomic lesions, the unidirectional conversion pathway was preferentially utilized even though the knock-in mutation could theoretically have been converted by both pathways. Collectively, our results suggest that ODN-mediated PGE utilizes synthesis-dependent strand annealing and single-stranded DNA incorporation pathways. Both of these pathways generate short conversion tracts with Gaussian-like distributions. Although synthesis-dependent strand annealing is preferentially utilized, our work unequivocally establishes the existence of a single-stranded DNA incorporation pathway in human cells. This work extends the paradigms of HDR-mediated gene conversion and establishes guidelines for PGE in human cells. Cold Spring Harbor Laboratory Press 2017-07 /pmc/articles/PMC5495063/ /pubmed/28356322 http://dx.doi.org/10.1101/gr.214775.116 Text en © 2017 Kan et al.; Published by Cold Spring Harbor Laboratory Press http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/. |
spellingShingle | Research Kan, Yinan Ruis, Brian Takasugi, Taylor Hendrickson, Eric A. Mechanisms of precise genome editing using oligonucleotide donors |
title | Mechanisms of precise genome editing using oligonucleotide donors |
title_full | Mechanisms of precise genome editing using oligonucleotide donors |
title_fullStr | Mechanisms of precise genome editing using oligonucleotide donors |
title_full_unstemmed | Mechanisms of precise genome editing using oligonucleotide donors |
title_short | Mechanisms of precise genome editing using oligonucleotide donors |
title_sort | mechanisms of precise genome editing using oligonucleotide donors |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495063/ https://www.ncbi.nlm.nih.gov/pubmed/28356322 http://dx.doi.org/10.1101/gr.214775.116 |
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