Tunable Repression of Key Photosynthetic Processes Using Cas12a CRISPR Interference in the Fast-Growing Cyanobacterium Synechococcus sp. UTEX 2973

Knoot, Cory J.; Biswas, Sandeep; Pakrasi, Himadri B.

doi:10.1021/acssynbio.9b00417

Title: Tunable Repression of Key Photosynthetic Processes Using Cas12a CRISPR Interference in the Fast-Growing Cyanobacterium Synechococcus sp. UTEX 2973

Journal Article · Thu Dec 12 00:00:00 EST 2019 · ACS Synthetic Biology

DOI:https://doi.org/10.1021/acssynbio.9b00417· OSTI ID:1800965

^[1]; Biswas, Sandeep ^[1];

^[1]

Washington Univ., St. Louis, MO (United States)

Cyanobacteria are photoautotrophic prokaryotes that serve as key model organisms to study basic photosynthetic processes and are potential carbon-negative production chassis for commodity and high-value chemicals. The development of new synthetic biology tools and improvement of current ones is a requisite for furthering these organisms as models and production vehicles. CRISPR interference (CRISPRi) allows for targeted gene repression using a DNase-dead Cas nuclease (“dCas”). Here, we describe a titratable dCas12a (dCpf1) CRISPRi system and apply it to repress key photosynthetic processes in the fast-growing cyanobacterium Synechococcus sp. UTEX 2973 (S2973). The system relies on a lac repressor system that retains tight regulation in the absence of inducer (0–10% repression) while maintaining the capability for >90% repression of high-abundance gene targets. We determined that dCas12a is less toxic than dCas9. We tested the efficacy of the system toward eYFP and three native targets in S2973: the phycobilisome antenna, glycogen synthesis, and photosystem I (PSI), an essential part of the photosynthetic electron transport chain in oxygenic photoautotrophs. PSI was knocked down indirectly by repressing the protein factor BtpA involved in stabilizing core PSI proteins. We could reduce cellular PSI titer by 87% under photoautotrophic conditions, and we characterized these cells to gain insights into the response of the strain to the low PSI content. The ability to tightly regulate and time the (de)repression of essential genes in trans will allow for the study of photosynthetic processes that are not accessible using knockout mutants.

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Research Organization:: Washington Univ., St. Louis, MO (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: FG02-99ER20350

OSTI ID:: 1800965

Journal Information:: ACS Synthetic Biology, Vol. 9, Issue 1; ISSN 2161-5063

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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Title: Tunable Repression of Key Photosynthetic Processes Using Cas12a CRISPR Interference in the Fast-Growing Cyanobacterium Synechococcus sp. UTEX 2973

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