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

Mihris Ibnu Saleem Naduthodi; Christian Südfeld; Emmanouil Klimis Avitzigiannis; Nicola Trevisan; Eduard van Lith; Javier Alcaide Sancho; Sarah D'Adamo; Maria Barbosa; John van der Oost

doi:10.1021/acssynbio.1c00329

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

Journal article

Open access

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

Mihris Ibnu Saleem Naduthodi, Christian Südfeld, Emmanouil Klimis Avitzigiannis, Nicola Trevisan, Eduard van Lith, Javier Alcaide Sancho, Sarah D'Adamo, Maria Barbosa and John van der Oost

ACS synthetic biology, Vol.10(12), pp.3369-3378

12/17/2021

DOI: 10.1021/acssynbio.1c00329

PMCID: PMC8689688

PMID: 34793143

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8689688View

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Abstract

Microalgae can produce industrially relevant metabolites using atmospheric CO 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 . 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 . Overall, these developments substantially accelerate genome engineering efforts in and potentially provide a general toolbox for improving other microalgal strains.

CRISPR-Cas Systems - genetics

Gene Editing

Genome

Microalgae - genetics

Stramenopiles - genetics

Details

Title: Subtitle: Comprehensive Genome Engineering Toolbox for Microalgae Nannochloropsis oceanica Based on CRISPR-Cas Systems
Creators: Mihris Ibnu Saleem Naduthodi - Wageningen University & Research
Christian Südfeld - Wageningen University & Research
Emmanouil Klimis Avitzigiannis - Wageningen University & Research
Nicola Trevisan - Wageningen University & Research
Eduard van Lith - Laboratory of Microbiology, Wageningen University, Wageningen 6708 WE, The Netherlands
Javier Alcaide Sancho - Laboratory of Microbiology, Wageningen University, Wageningen 6708 WE, The Netherlands
Sarah D'Adamo - Wageningen University & Research
Maria Barbosa - Wageningen University & Research
John van der Oost - Wageningen University & Research
Resource Type: Journal article
Publication Details: ACS synthetic biology, Vol.10(12), pp.3369-3378
DOI: 10.1021/acssynbio.1c00329
PMID: 34793143
PMCID: PMC8689688
NLM abbreviation: ACS Synth Biol
ISSN: 2161-5063
eISSN: 2161-5063
Grant note: DOI: 10.13039/100010661, name: Horizon 2020 Framework Programme, award: 745754
Language: English
Date published: 12/17/2021
Academic Unit: International Programs; Spanish and Portuguese
Record Identifier: 9984398035702771

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