Journal
ACS SYNTHETIC BIOLOGY
Volume 9, Issue 9, Pages 2351-2361Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.0c00091
Keywords
cyanobacteria; CRISPR interference; Cas12a; squalene; synthetic biology
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Funding
- Individual Research program through the National Research Foundation of Korea - Korean Government (Ministry of science an ICT) [2017R1A2B2002566, 2018R1D1A1B07043967]
- Technology Innovation Program - Ministry of Trade, Industry and Energy [20000158]
- National Research Foundation of Korea [2018R1D1A1B07043967] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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In cyanobacteria, metabolic engineering using synthetic biology tools is limited to build a biosolar cell factory that converts CO2 to value-added chemicals, as repression of essential genes has not been achieved. In this study, we developed a dCas12a-mediated CRISPR interference system (CRISPRi-dCas12a) in cyanobacteria that effectively blocked the transcriptional initiation by means of a CRISPR-RNA (crRNA) and 19-nt direct repeat, resulting in 53-94% gene repression. The repression of multiple genes in a single crRNA array was also successfully achieved without a loss in repression strength. In addition, as a demonstration of the dCas12a-mediated CRISPRi for metabolic engineering, photosynthetic squalene production was improved by repressing the essential genes of either acnB encoding for aconitase or cpcB(2) encoding for phycocyanin beta-subunit in Synechococcus elongatus PCC 7942. The ability to regulate gene repression will promote the construction of biosolar cell factories to produce valueadded chemicals.
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