Cargando…

Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in Vivo Genome Editing

Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases. Genome editing represents a promising emerging field in the treatment of monogenic disorders, as it aims to correct disease-causing mutations within the genome. Genome editing relies on highly spec...

Descripción completa

Detalles Bibliográficos
Autores principales: Pasquini, Giovanni, Cora, Virginia, Swiersy, Anka, Achberger, Kevin, Antkowiak, Lena, Müller, Brigitte, Wimmer, Tobias, Fraschka, Sabine Anne-Kristin, Casadei, Nicolas, Ueffing, Marius, Liebau, Stefan, Stieger, Knut, Busskamp, Volker
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073035/
https://www.ncbi.nlm.nih.gov/pubmed/32085662
http://dx.doi.org/10.3390/ijms21041380
_version_ 1783506544692822016
author Pasquini, Giovanni
Cora, Virginia
Swiersy, Anka
Achberger, Kevin
Antkowiak, Lena
Müller, Brigitte
Wimmer, Tobias
Fraschka, Sabine Anne-Kristin
Casadei, Nicolas
Ueffing, Marius
Liebau, Stefan
Stieger, Knut
Busskamp, Volker
author_facet Pasquini, Giovanni
Cora, Virginia
Swiersy, Anka
Achberger, Kevin
Antkowiak, Lena
Müller, Brigitte
Wimmer, Tobias
Fraschka, Sabine Anne-Kristin
Casadei, Nicolas
Ueffing, Marius
Liebau, Stefan
Stieger, Knut
Busskamp, Volker
author_sort Pasquini, Giovanni
collection PubMed
description Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases. Genome editing represents a promising emerging field in the treatment of monogenic disorders, as it aims to correct disease-causing mutations within the genome. Genome editing relies on highly specific endonucleases and the capacity of the cells to repair double-strand breaks (DSBs). As DSB pathways are cell-cycle dependent, their activity in postmitotic retinal neurons, with a focus on photoreceptors, needs to be assessed in order to develop therapeutic in vivo genome editing. Three DSB-repair pathways are found in mammalian cells: Non-homologous end joining (NHEJ); microhomology-mediated end joining (MMEJ); and homology-directed repair (HDR). While NHEJ can be used to knock out mutant alleles in dominant disorders, HDR and MMEJ are better suited for precise genome editing, or for replacing entire mutation hotspots in genomic regions. Here, we analyzed transcriptomic in vivo and in vitro data and revealed that HDR is indeed downregulated in postmitotic neurons, whereas MMEJ and NHEJ are active. Using single-cell RNA sequencing analysis, we characterized the dynamics of DSB repair pathways in the transition from dividing cells to postmitotic retinal cells. Time-course bulk RNA-seq data confirmed DSB repair gene expression in both in vivo and in vitro samples. Transcriptomic DSB repair pathway profiles are very similar in adult human, macaque, and mouse retinas, but not in ground squirrel retinas. Moreover, human-induced pluripotent stem-cell-derived neurons and retinal organoids can serve as well suited in vitro testbeds for developing genomic engineering approaches in photoreceptors. Our study provides additional support for designing precise in vivo genome-editing approaches via MMEJ, which is active in mature photoreceptors.
format Online
Article
Text
id pubmed-7073035
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-70730352020-03-19 Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in Vivo Genome Editing Pasquini, Giovanni Cora, Virginia Swiersy, Anka Achberger, Kevin Antkowiak, Lena Müller, Brigitte Wimmer, Tobias Fraschka, Sabine Anne-Kristin Casadei, Nicolas Ueffing, Marius Liebau, Stefan Stieger, Knut Busskamp, Volker Int J Mol Sci Article Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases. Genome editing represents a promising emerging field in the treatment of monogenic disorders, as it aims to correct disease-causing mutations within the genome. Genome editing relies on highly specific endonucleases and the capacity of the cells to repair double-strand breaks (DSBs). As DSB pathways are cell-cycle dependent, their activity in postmitotic retinal neurons, with a focus on photoreceptors, needs to be assessed in order to develop therapeutic in vivo genome editing. Three DSB-repair pathways are found in mammalian cells: Non-homologous end joining (NHEJ); microhomology-mediated end joining (MMEJ); and homology-directed repair (HDR). While NHEJ can be used to knock out mutant alleles in dominant disorders, HDR and MMEJ are better suited for precise genome editing, or for replacing entire mutation hotspots in genomic regions. Here, we analyzed transcriptomic in vivo and in vitro data and revealed that HDR is indeed downregulated in postmitotic neurons, whereas MMEJ and NHEJ are active. Using single-cell RNA sequencing analysis, we characterized the dynamics of DSB repair pathways in the transition from dividing cells to postmitotic retinal cells. Time-course bulk RNA-seq data confirmed DSB repair gene expression in both in vivo and in vitro samples. Transcriptomic DSB repair pathway profiles are very similar in adult human, macaque, and mouse retinas, but not in ground squirrel retinas. Moreover, human-induced pluripotent stem-cell-derived neurons and retinal organoids can serve as well suited in vitro testbeds for developing genomic engineering approaches in photoreceptors. Our study provides additional support for designing precise in vivo genome-editing approaches via MMEJ, which is active in mature photoreceptors. MDPI 2020-02-18 /pmc/articles/PMC7073035/ /pubmed/32085662 http://dx.doi.org/10.3390/ijms21041380 Text en © 2020 by the authors. 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 (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Pasquini, Giovanni
Cora, Virginia
Swiersy, Anka
Achberger, Kevin
Antkowiak, Lena
Müller, Brigitte
Wimmer, Tobias
Fraschka, Sabine Anne-Kristin
Casadei, Nicolas
Ueffing, Marius
Liebau, Stefan
Stieger, Knut
Busskamp, Volker
Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in Vivo Genome Editing
title Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in Vivo Genome Editing
title_full Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in Vivo Genome Editing
title_fullStr Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in Vivo Genome Editing
title_full_unstemmed Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in Vivo Genome Editing
title_short Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in Vivo Genome Editing
title_sort using transcriptomic analysis to assess double-strand break repair activity: towards precise in vivo genome editing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073035/
https://www.ncbi.nlm.nih.gov/pubmed/32085662
http://dx.doi.org/10.3390/ijms21041380
work_keys_str_mv AT pasquinigiovanni usingtranscriptomicanalysistoassessdoublestrandbreakrepairactivitytowardspreciseinvivogenomeediting
AT coravirginia usingtranscriptomicanalysistoassessdoublestrandbreakrepairactivitytowardspreciseinvivogenomeediting
AT swiersyanka usingtranscriptomicanalysistoassessdoublestrandbreakrepairactivitytowardspreciseinvivogenomeediting
AT achbergerkevin usingtranscriptomicanalysistoassessdoublestrandbreakrepairactivitytowardspreciseinvivogenomeediting
AT antkowiaklena usingtranscriptomicanalysistoassessdoublestrandbreakrepairactivitytowardspreciseinvivogenomeediting
AT mullerbrigitte usingtranscriptomicanalysistoassessdoublestrandbreakrepairactivitytowardspreciseinvivogenomeediting
AT wimmertobias usingtranscriptomicanalysistoassessdoublestrandbreakrepairactivitytowardspreciseinvivogenomeediting
AT fraschkasabineannekristin usingtranscriptomicanalysistoassessdoublestrandbreakrepairactivitytowardspreciseinvivogenomeediting
AT casadeinicolas usingtranscriptomicanalysistoassessdoublestrandbreakrepairactivitytowardspreciseinvivogenomeediting
AT ueffingmarius usingtranscriptomicanalysistoassessdoublestrandbreakrepairactivitytowardspreciseinvivogenomeediting
AT liebaustefan usingtranscriptomicanalysistoassessdoublestrandbreakrepairactivitytowardspreciseinvivogenomeediting
AT stiegerknut usingtranscriptomicanalysistoassessdoublestrandbreakrepairactivitytowardspreciseinvivogenomeediting
AT busskampvolker usingtranscriptomicanalysistoassessdoublestrandbreakrepairactivitytowardspreciseinvivogenomeediting