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CRISPR‐Cas9‐mediated efficient directed mutagenesis and RAD51‐dependent and RAD51‐independent gene targeting in the moss Physcomitrella patens

The ability to address the CRISPR‐Cas9 nuclease complex to any target DNA using customizable single‐guide RNAs has now permitted genome engineering in many species. Here, we report its first successful use in a nonvascular plant, the moss Physcomitrella patens. Single‐guide RNAs (sgRNAs) were design...

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Autores principales: Collonnier, Cécile, Epert, Aline, Mara, Kostlend, Maclot, François, Guyon‐Debast, Anouchka, Charlot, Florence, White, Charles, Schaefer, Didier G., Nogué, Fabien
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5253467/
https://www.ncbi.nlm.nih.gov/pubmed/27368642
http://dx.doi.org/10.1111/pbi.12596
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author Collonnier, Cécile
Epert, Aline
Mara, Kostlend
Maclot, François
Guyon‐Debast, Anouchka
Charlot, Florence
White, Charles
Schaefer, Didier G.
Nogué, Fabien
author_facet Collonnier, Cécile
Epert, Aline
Mara, Kostlend
Maclot, François
Guyon‐Debast, Anouchka
Charlot, Florence
White, Charles
Schaefer, Didier G.
Nogué, Fabien
author_sort Collonnier, Cécile
collection PubMed
description The ability to address the CRISPR‐Cas9 nuclease complex to any target DNA using customizable single‐guide RNAs has now permitted genome engineering in many species. Here, we report its first successful use in a nonvascular plant, the moss Physcomitrella patens. Single‐guide RNAs (sgRNAs) were designed to target an endogenous reporter gene, PpAPT, whose inactivation confers resistance to 2‐fluoroadenine. Transformation of moss protoplasts with these sgRNAs and the Cas9 coding sequence from Streptococcus pyogenes triggered mutagenesis at the PpAPT target in about 2% of the regenerated plants. Mainly, deletions were observed, most of them resulting from alternative end‐joining (alt‐EJ)‐driven repair. We further demonstrate that, in the presence of a donor DNA sharing sequence homology with the PpAPT gene, most transgene integration events occur by homology‐driven repair (HDR) at the target locus but also that Cas9‐induced double‐strand breaks are repaired with almost equal frequencies by mutagenic illegitimate recombination. Finally, we establish that a significant fraction of HDR‐mediated gene targeting events (30%) is still possible in the absence of PpRAD51 protein, indicating that CRISPR‐induced HDR is only partially mediated by the classical homologous recombination pathway.
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spelling pubmed-52534672017-02-03 CRISPR‐Cas9‐mediated efficient directed mutagenesis and RAD51‐dependent and RAD51‐independent gene targeting in the moss Physcomitrella patens Collonnier, Cécile Epert, Aline Mara, Kostlend Maclot, François Guyon‐Debast, Anouchka Charlot, Florence White, Charles Schaefer, Didier G. Nogué, Fabien Plant Biotechnol J Research Articles The ability to address the CRISPR‐Cas9 nuclease complex to any target DNA using customizable single‐guide RNAs has now permitted genome engineering in many species. Here, we report its first successful use in a nonvascular plant, the moss Physcomitrella patens. Single‐guide RNAs (sgRNAs) were designed to target an endogenous reporter gene, PpAPT, whose inactivation confers resistance to 2‐fluoroadenine. Transformation of moss protoplasts with these sgRNAs and the Cas9 coding sequence from Streptococcus pyogenes triggered mutagenesis at the PpAPT target in about 2% of the regenerated plants. Mainly, deletions were observed, most of them resulting from alternative end‐joining (alt‐EJ)‐driven repair. We further demonstrate that, in the presence of a donor DNA sharing sequence homology with the PpAPT gene, most transgene integration events occur by homology‐driven repair (HDR) at the target locus but also that Cas9‐induced double‐strand breaks are repaired with almost equal frequencies by mutagenic illegitimate recombination. Finally, we establish that a significant fraction of HDR‐mediated gene targeting events (30%) is still possible in the absence of PpRAD51 protein, indicating that CRISPR‐induced HDR is only partially mediated by the classical homologous recombination pathway. John Wiley and Sons Inc. 2016-07-22 2017-01 /pmc/articles/PMC5253467/ /pubmed/27368642 http://dx.doi.org/10.1111/pbi.12596 Text en © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution (http://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Articles
Collonnier, Cécile
Epert, Aline
Mara, Kostlend
Maclot, François
Guyon‐Debast, Anouchka
Charlot, Florence
White, Charles
Schaefer, Didier G.
Nogué, Fabien
CRISPR‐Cas9‐mediated efficient directed mutagenesis and RAD51‐dependent and RAD51‐independent gene targeting in the moss Physcomitrella patens
title CRISPR‐Cas9‐mediated efficient directed mutagenesis and RAD51‐dependent and RAD51‐independent gene targeting in the moss Physcomitrella patens
title_full CRISPR‐Cas9‐mediated efficient directed mutagenesis and RAD51‐dependent and RAD51‐independent gene targeting in the moss Physcomitrella patens
title_fullStr CRISPR‐Cas9‐mediated efficient directed mutagenesis and RAD51‐dependent and RAD51‐independent gene targeting in the moss Physcomitrella patens
title_full_unstemmed CRISPR‐Cas9‐mediated efficient directed mutagenesis and RAD51‐dependent and RAD51‐independent gene targeting in the moss Physcomitrella patens
title_short CRISPR‐Cas9‐mediated efficient directed mutagenesis and RAD51‐dependent and RAD51‐independent gene targeting in the moss Physcomitrella patens
title_sort crispr‐cas9‐mediated efficient directed mutagenesis and rad51‐dependent and rad51‐independent gene targeting in the moss physcomitrella patens
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5253467/
https://www.ncbi.nlm.nih.gov/pubmed/27368642
http://dx.doi.org/10.1111/pbi.12596
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