TY - JOUR
T1 - A Reversibly Induced CRISPRi System Targeting Photosystem II in the Cyanobacterium Synechocystis sp. PCC 6803
AU - Liu, Deng
AU - Johnson, Virginia M.
AU - Pakrasi, Himadri B.
N1 - Funding Information:
We thank members of the Pakrasi lab for congenial discussion. This work was supported by the Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (DOE) (Grant DE-FG02-99ER20350 to H.B.P.). V.M.J was supported by a training grant T32 EB014855 from the National Institute of Biomedical Imaging and Bioengineering, NIH.
Publisher Copyright:
Copyright © 2020 American Chemical Society.
PY - 2020/6/19
Y1 - 2020/6/19
N2 - The cyanobacterium Synechocystis sp. PCC 6803 is used as a model organism to study photosynthesis, as it can utilize glucose as the sole carbon source to support its growth under heterotrophic conditions. CRISPR interference (CRISPRi) has been widely applied to repress the transcription of genes in a targeted manner in cyanobacteria. However, a robust and reversible induced CRISPRi system has not been explored in Synechocystis 6803 to knock down and recover the expression of a targeted gene. In this study, we built a tightly controlled chimeric promoter, PrhaBAD-RSW, in which a theophylline responsive riboswitch was integrated into a rhamnose-inducible promoter system. We applied this promoter to drive the expression of ddCpf1 (DNase-dead Cpf1 nuclease) in a CRISPRi system and chose the PSII reaction center gene psbD (D2 protein) to target for repression. psbD was specifically knocked down by over 95% of its native expression, leading to severely inhibited photosystem II activity and growth of Synechocystis 6803 under photoautotrophic conditions. Significantly, removal of the inducers rhamnose and theophylline reversed repression by CRISPRi. Expression of PsbD recovered following release of repression, coupled with increased photosystem II content and activity. This reversibly induced CRISPRi system in Synechocystis 6803 represents a new strategy for study of the biogenesis of photosynthetic complexes in cyanobacteria.
AB - The cyanobacterium Synechocystis sp. PCC 6803 is used as a model organism to study photosynthesis, as it can utilize glucose as the sole carbon source to support its growth under heterotrophic conditions. CRISPR interference (CRISPRi) has been widely applied to repress the transcription of genes in a targeted manner in cyanobacteria. However, a robust and reversible induced CRISPRi system has not been explored in Synechocystis 6803 to knock down and recover the expression of a targeted gene. In this study, we built a tightly controlled chimeric promoter, PrhaBAD-RSW, in which a theophylline responsive riboswitch was integrated into a rhamnose-inducible promoter system. We applied this promoter to drive the expression of ddCpf1 (DNase-dead Cpf1 nuclease) in a CRISPRi system and chose the PSII reaction center gene psbD (D2 protein) to target for repression. psbD was specifically knocked down by over 95% of its native expression, leading to severely inhibited photosystem II activity and growth of Synechocystis 6803 under photoautotrophic conditions. Significantly, removal of the inducers rhamnose and theophylline reversed repression by CRISPRi. Expression of PsbD recovered following release of repression, coupled with increased photosystem II content and activity. This reversibly induced CRISPRi system in Synechocystis 6803 represents a new strategy for study of the biogenesis of photosynthetic complexes in cyanobacteria.
KW - CRISPR interference
KW - cyanobacteria
KW - photosystem II
UR - http://www.scopus.com/inward/record.url?scp=85086749175&partnerID=8YFLogxK
U2 - 10.1021/acssynbio.0c00106
DO - 10.1021/acssynbio.0c00106
M3 - Article
C2 - 32379958
AN - SCOPUS:85086749175
SN - 2161-5063
VL - 9
SP - 1441
EP - 1449
JO - ACS synthetic biology
JF - ACS synthetic biology
IS - 6
ER -