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Systematic analysis of factors that improve homologous direct repair (HDR) efficiency in CRISPR/Cas9 technique

The CRISPR/Cas9 bacterial system has proven to be an powerful tool for genetic manipulation in several organisms, but the efficiency of sequence replacement by homologous direct repair (HDR) is substantially lower than random indel creation. Many studies focused on improving HDR efficiency using dou...

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Detalles Bibliográficos
Autores principales: Di Stazio, Mariateresa, Foschi, Nicola, Athanasakis, Emmanouil, Gasparini, Paolo, d’Adamo, Adamo Pio
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935300/
https://www.ncbi.nlm.nih.gov/pubmed/33667229
http://dx.doi.org/10.1371/journal.pone.0247603
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author Di Stazio, Mariateresa
Foschi, Nicola
Athanasakis, Emmanouil
Gasparini, Paolo
d’Adamo, Adamo Pio
author_facet Di Stazio, Mariateresa
Foschi, Nicola
Athanasakis, Emmanouil
Gasparini, Paolo
d’Adamo, Adamo Pio
author_sort Di Stazio, Mariateresa
collection PubMed
description The CRISPR/Cas9 bacterial system has proven to be an powerful tool for genetic manipulation in several organisms, but the efficiency of sequence replacement by homologous direct repair (HDR) is substantially lower than random indel creation. Many studies focused on improving HDR efficiency using double sgRNA, cell synchronization cycle, and the delivery of single-stranded oligo DNA nucleotides (ssODN) with a rational design. In this study, we evaluate these three methods’ synergistic effects to improve HDR efficiency. For our tests, we have chosen the TNFα gene (NM_000594) for its crucial role in various biological processes and diseases. For the first time, our results showed how the use of two sgRNA with asymmetric donor design and triple transfection events dramatically increase the HDR efficiency from an undetectable HDR event to 39% of HDR efficiency and provide a new strategy to facilitate CRISPR/Cas9-mediated human genome editing. Besides, we demonstrated that the TNFα locus could be edited with CRISPR/Cas9 methodology, an opportunity to safely correct, in the future, the specific mutations of each patient.
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spelling pubmed-79353002021-03-15 Systematic analysis of factors that improve homologous direct repair (HDR) efficiency in CRISPR/Cas9 technique Di Stazio, Mariateresa Foschi, Nicola Athanasakis, Emmanouil Gasparini, Paolo d’Adamo, Adamo Pio PLoS One Research Article The CRISPR/Cas9 bacterial system has proven to be an powerful tool for genetic manipulation in several organisms, but the efficiency of sequence replacement by homologous direct repair (HDR) is substantially lower than random indel creation. Many studies focused on improving HDR efficiency using double sgRNA, cell synchronization cycle, and the delivery of single-stranded oligo DNA nucleotides (ssODN) with a rational design. In this study, we evaluate these three methods’ synergistic effects to improve HDR efficiency. For our tests, we have chosen the TNFα gene (NM_000594) for its crucial role in various biological processes and diseases. For the first time, our results showed how the use of two sgRNA with asymmetric donor design and triple transfection events dramatically increase the HDR efficiency from an undetectable HDR event to 39% of HDR efficiency and provide a new strategy to facilitate CRISPR/Cas9-mediated human genome editing. Besides, we demonstrated that the TNFα locus could be edited with CRISPR/Cas9 methodology, an opportunity to safely correct, in the future, the specific mutations of each patient. Public Library of Science 2021-03-05 /pmc/articles/PMC7935300/ /pubmed/33667229 http://dx.doi.org/10.1371/journal.pone.0247603 Text en © 2021 Di Stazio et al 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 use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Di Stazio, Mariateresa
Foschi, Nicola
Athanasakis, Emmanouil
Gasparini, Paolo
d’Adamo, Adamo Pio
Systematic analysis of factors that improve homologous direct repair (HDR) efficiency in CRISPR/Cas9 technique
title Systematic analysis of factors that improve homologous direct repair (HDR) efficiency in CRISPR/Cas9 technique
title_full Systematic analysis of factors that improve homologous direct repair (HDR) efficiency in CRISPR/Cas9 technique
title_fullStr Systematic analysis of factors that improve homologous direct repair (HDR) efficiency in CRISPR/Cas9 technique
title_full_unstemmed Systematic analysis of factors that improve homologous direct repair (HDR) efficiency in CRISPR/Cas9 technique
title_short Systematic analysis of factors that improve homologous direct repair (HDR) efficiency in CRISPR/Cas9 technique
title_sort systematic analysis of factors that improve homologous direct repair (hdr) efficiency in crispr/cas9 technique
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935300/
https://www.ncbi.nlm.nih.gov/pubmed/33667229
http://dx.doi.org/10.1371/journal.pone.0247603
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