Application of CRISPRi technology to unravel the biological role of the archaeal LonB protease

MARCHFELDER, A; FERRARI, M.C.; DE CASTRO, R; SCHWARZ, T.S.

Les Diablerets

Conferencia; Archaea: Ecology, Metabolism and Molecular Biology Gordon Research Conference; 2019

Archaea: Ecology, Metabolism and Molecular Biology - Gordon Research Conference

ATP-dependent Lon proteases are conserved among the three domains of life. In Archaea this enzyme is membrane-associated and its biological relevance and natural targets are almost known. To address this issue, we applied the pop-in pop-out method to obtain mutant strains deficient in the lonB gene in the model haloarchaeon Haloferax volcanii. lonB was essential for cell viability, thus, a conditional LonB mutant (HVLON3) was constructed introducing the ptna promoter upstream the lon gene by homologous recombination. This strain was affected in growth rate, cell shape and produced hyperpigmented colonies under reduced Lon expression. Applying various proteomics strategies combined with MS analysis to HVLON3, potential LonB targets were identified, including phytoene synthase and Cas7 (Cerletti et al J. Proteome Res 2018). The expression of the recombinant targets under the ptna promoter in the genetic background of HVLON3 was not possible. Thus, an alternative genetic tool was necessary to validate the LonB targets. Since the H. volcanii CRISPR-Cas system has been harnessed as a tool to repress gene expression in this haloarchaeon (CRISPRi), the aim of this work was to apply CRISPRi to silence lonB expression in H. volcanii to facilitate the study of the biological function of this protease in archaeal cells and to validate its potential target proteins.Three crRNAs were generated targeting the promoter region and/or the transcription initiation site of the lonB gene (HVO_0783). Haloferax cells were transformed with these crRNAs and examined phenotypically, showing that all three crRNAs produced hyperpigmented colonies. To evaluate targeting efficiency of the different crRNAs, cell growth and carotenoid content were determined for each strain. Cells expressing the crRNA#antiLonB1 showed a growth defect and produced hyperpigmented colonies. Total RNA was isolated from the different strains and analyzed by Northern analyses to verify lonB repression. As expected, the crRNA #antiLonB1 had the strongest effect on lonB expression (67% inhibition) which was consistent with the phenotype of the silenced strain. This work shows the successful silencing of the lonB protease gene in H. volcanii by CRISPRi, providing a suitable host strain for the expression of recombinant proteins.