Cargando…
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...
Autores principales: | , , , , , , , , |
---|---|
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 |
_version_ | 1784618587348008960 |
---|---|
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. |
format | Online Article Text |
id | pubmed-8689688 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT naduthodimihrisibnusaleem comprehensivegenomeengineeringtoolboxformicroalgaenannochloropsisoceanicabasedoncrisprcassystems AT sudfeldchristian comprehensivegenomeengineeringtoolboxformicroalgaenannochloropsisoceanicabasedoncrisprcassystems AT avitzigiannisemmanouilklimis comprehensivegenomeengineeringtoolboxformicroalgaenannochloropsisoceanicabasedoncrisprcassystems AT trevisannicola comprehensivegenomeengineeringtoolboxformicroalgaenannochloropsisoceanicabasedoncrisprcassystems AT vanlitheduard comprehensivegenomeengineeringtoolboxformicroalgaenannochloropsisoceanicabasedoncrisprcassystems AT alcaidesanchojavier comprehensivegenomeengineeringtoolboxformicroalgaenannochloropsisoceanicabasedoncrisprcassystems AT dadamosarah comprehensivegenomeengineeringtoolboxformicroalgaenannochloropsisoceanicabasedoncrisprcassystems AT barbosamaria comprehensivegenomeengineeringtoolboxformicroalgaenannochloropsisoceanicabasedoncrisprcassystems AT vanderoostjohn comprehensivegenomeengineeringtoolboxformicroalgaenannochloropsisoceanicabasedoncrisprcassystems |