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Comprehensive Genome Engineering Toolbox for Microalgae Nannochloropsis oceanica Based on CRISPR-Cas Systems

[Image: see text] Microalgae can produce industrially relevant metabolites using atmospheric CO(2) and sunlight as carbon and energy sources, respectively. Developing molecular tools for high-throughput genome engineering could accelerate the generation of tailored strains with improved traits. To t...

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Autores principales: Naduthodi, Mihris Ibnu Saleem, Südfeld, Christian, Avitzigiannis, Emmanouil Klimis, Trevisan, Nicola, van Lith, Eduard, Alcaide Sancho, Javier, D’Adamo, Sarah, Barbosa, Maria, van der Oost, John
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2021
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8689688/
https://www.ncbi.nlm.nih.gov/pubmed/34793143
http://dx.doi.org/10.1021/acssynbio.1c00329
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author Naduthodi, Mihris Ibnu Saleem
Südfeld, Christian
Avitzigiannis, Emmanouil Klimis
Trevisan, Nicola
van Lith, Eduard
Alcaide Sancho, Javier
D’Adamo, Sarah
Barbosa, Maria
van der Oost, John
author_facet Naduthodi, Mihris Ibnu Saleem
Südfeld, Christian
Avitzigiannis, Emmanouil Klimis
Trevisan, Nicola
van Lith, Eduard
Alcaide Sancho, Javier
D’Adamo, Sarah
Barbosa, Maria
van der Oost, John
author_sort Naduthodi, Mihris Ibnu Saleem
collection PubMed
description [Image: see text] Microalgae can produce industrially relevant metabolites using atmospheric CO(2) and sunlight as carbon and energy sources, respectively. Developing molecular tools for high-throughput genome engineering could accelerate the generation of tailored strains with improved traits. To this end, we developed a genome editing strategy based on Cas12a ribonucleoproteins (RNPs) and homology-directed repair (HDR) to generate scarless and markerless mutants of the microalga Nannochloropsis oceanica. We also developed an episomal plasmid-based Cas12a system for efficiently introducing indels at the target site. Additionally, we exploited the ability of Cas12a to process an associated CRISPR array to perform multiplexed genome engineering. We efficiently targeted three sites in the host genome in a single transformation, thereby making a major step toward high-throughput genome engineering in microalgae. Furthermore, a CRISPR interference (CRISPRi) tool based on Cas9 and Cas12a was developed for effective downregulation of target genes. We observed up to 85% reduction in the transcript levels upon performing CRISPRi with dCas9 in N. oceanica. Overall, these developments substantially accelerate genome engineering efforts in N. oceanica and potentially provide a general toolbox for improving other microalgal strains.
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spelling pubmed-86896882021-12-22 Comprehensive Genome Engineering Toolbox for Microalgae Nannochloropsis oceanica Based on CRISPR-Cas Systems Naduthodi, Mihris Ibnu Saleem Südfeld, Christian Avitzigiannis, Emmanouil Klimis Trevisan, Nicola van Lith, Eduard Alcaide Sancho, Javier D’Adamo, Sarah Barbosa, Maria van der Oost, John ACS Synth Biol [Image: see text] Microalgae can produce industrially relevant metabolites using atmospheric CO(2) and sunlight as carbon and energy sources, respectively. Developing molecular tools for high-throughput genome engineering could accelerate the generation of tailored strains with improved traits. To this end, we developed a genome editing strategy based on Cas12a ribonucleoproteins (RNPs) and homology-directed repair (HDR) to generate scarless and markerless mutants of the microalga Nannochloropsis oceanica. We also developed an episomal plasmid-based Cas12a system for efficiently introducing indels at the target site. Additionally, we exploited the ability of Cas12a to process an associated CRISPR array to perform multiplexed genome engineering. We efficiently targeted three sites in the host genome in a single transformation, thereby making a major step toward high-throughput genome engineering in microalgae. Furthermore, a CRISPR interference (CRISPRi) tool based on Cas9 and Cas12a was developed for effective downregulation of target genes. We observed up to 85% reduction in the transcript levels upon performing CRISPRi with dCas9 in N. oceanica. Overall, these developments substantially accelerate genome engineering efforts in N. oceanica and potentially provide a general toolbox for improving other microalgal strains. American Chemical Society 2021-11-18 2021-12-17 /pmc/articles/PMC8689688/ /pubmed/34793143 http://dx.doi.org/10.1021/acssynbio.1c00329 Text en © 2021 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Naduthodi, Mihris Ibnu Saleem
Südfeld, Christian
Avitzigiannis, Emmanouil Klimis
Trevisan, Nicola
van Lith, Eduard
Alcaide Sancho, Javier
D’Adamo, Sarah
Barbosa, Maria
van der Oost, John
Comprehensive Genome Engineering Toolbox for Microalgae Nannochloropsis oceanica Based on CRISPR-Cas Systems
title Comprehensive Genome Engineering Toolbox for Microalgae Nannochloropsis oceanica Based on CRISPR-Cas Systems
title_full Comprehensive Genome Engineering Toolbox for Microalgae Nannochloropsis oceanica Based on CRISPR-Cas Systems
title_fullStr Comprehensive Genome Engineering Toolbox for Microalgae Nannochloropsis oceanica Based on CRISPR-Cas Systems
title_full_unstemmed Comprehensive Genome Engineering Toolbox for Microalgae Nannochloropsis oceanica Based on CRISPR-Cas Systems
title_short Comprehensive Genome Engineering Toolbox for Microalgae Nannochloropsis oceanica Based on CRISPR-Cas Systems
title_sort comprehensive genome engineering toolbox for microalgae nannochloropsis oceanica based on crispr-cas systems
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8689688/
https://www.ncbi.nlm.nih.gov/pubmed/34793143
http://dx.doi.org/10.1021/acssynbio.1c00329
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