Safe-harboring based novel genetic toolkit for Nannochloropsis salina CCMP1776: efficient overexpression of transgene via CRISPR/Cas9-mediated Knock-in at the transcriptional hotspot

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Title
Safe-harboring based novel genetic toolkit for Nannochloropsis salina CCMP1776: efficient overexpression of transgene via CRISPR/Cas9-mediated Knock-in at the transcriptional hotspot
Author(s)
A J Ryu; B R Jeong; N K Kang; S Jeon; M G Sohn; H J Yun; Jong Min Lim; S W Jeong; Y I Park; Won Joong Jeong; S Park; Y K Chang; K J Jeong
Bibliographic Citation
Bioresource Technology, vol. 340, pp. 125676-125676
Publication Year
2021
Abstract
Transgene expression in microalgae can be hampered by transgene silencing and unstable expression due to position effects. To overcome this, “safe harboring” transgene expression system was established for Nannochloropsis. Initially, transformants were obtained expressing a sfGFP reporter, followed by screening for high expression of sfGFP with fluorescence-activated cell sorter (FACS). 'T1' transcriptional hotspot was identified from a mutant showing best expression of sfGFP, but did not affect growth or lipid contents. By using a Cas9 editor strain, FAD12 gene, encoding Δ12-fatty acid desaturase (FAD12), was successfully knocked-in at the T1 locus, resulting in significantly higher expression of FAD12 than those of random integration. Importantly, the “safe harbored” FAD12 transformants showed four-fold higher production of linoleic acid (LA), the product of FAD12, leading to 1.5-fold increase in eicosapentaenoic acid (EPA). This safe harboring principle provide excellent proof of the concept for successful genetic/metabolic engineering of microalgae and other organisms.
Keyword
CRISPR/Cas9Safe harboringNannochloropsis salinaFatty acid desaturaseKnock-in via NHEJ
ISSN
0960-8524
Publisher
Elsevier
DOI
http://dx.doi.org/10.1016/j.biortech.2021.125676
Type
Article
Appears in Collections:
Synthetic Biology and Bioengineering Research Institute > Cell Factory Research Center > 1. Journal Articles
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