A single plasmid based CRISPR interference in Synechocystis 6803 – A proof of concept

Kirtania, Prithwiraj; Hódi, Barbara; Mallick, Ivy; Vass, István Zoltán; Fehér, Tamás; Vass, Imre; Kós, Péter B.

doi:10.1371/journal.pone.0225375

Cited by 18 publications

(9 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As mentioned by Yao et al [17], the blocking of transcription elongation may not be as efficient if the gene of interest is part of an operon with a distant TSS. Nevertheless, the repression levels obtained in this study are within those previously reported for other targets in Synechocystis [17,26,27]. On the one hand, it is also possible to purposefully design the sgRNA further from the TSS, resulting in lower repression of the target gene(s) and enabling fine-tuning gene expression, as demonstrated by Shabestary et al [26].…”

Section: Repression Of Three Kpsm Homologues In Synechocystis and Characterization Of The 3-sgrna Mutantsupporting

confidence: 84%

CRISPRi as a Tool to Repress Multiple Copies of Extracellular Polymeric Substances (EPS)-Related Genes in the Cyanobacterium Synechocystis sp. PCC 6803

Santos

Pacheco

Yao

et al. 2021

Life

View full text Add to dashboard Cite

The use of the versatile cyanobacterial extracellular polymeric substances (EPS) for biotechnological/biomedical applications implies an extensive knowledge of their biosynthetic pathways to improve/control polymer production yields and characteristics. The multiple copies of EPS-related genes, scattered throughout cyanobacterial genomes, adds another layer of complexity, making these studies challenging and time-consuming. Usually, this issue would be tackled by generating deletion mutants, a process that in cyanobacteria is also hindered by the polyploidy. Thus, the use of the CRISPRi multiplex system constitutes an efficient approach to addressing this redundancy. Here, three putative Synechocystis sp. PCC 6803 kpsM homologues (slr0977, slr2107, and sll0574) were repressed using this methodology. The characterization of the 3-sgRNA mutant in terms of fitness/growth and total carbohydrates, released and capsular polysaccharides, and its comparison with previously generated single knockout mutants pointed towards Slr0977 being the key KpsM player in Synechocystis EPS production. This work validates CRISPRi as a powerful tool to unravel cyanobacterial complex EPS biosynthetic pathways expediting this type of studies.

show abstract

Section: Repression Of Three Kpsm Homologues In Synechocystis and Characterization Of The 3-sgrna Mutantsupporting

confidence: 84%

CRISPRi as a Tool to Repress Multiple Copies of Extracellular Polymeric Substances (EPS)-Related Genes in the Cyanobacterium Synechocystis sp. PCC 6803

Santos

Pacheco

Yao

et al. 2021

Life

View full text Add to dashboard Cite

show abstract

“…In one example of this type of strategy, DNase-dead Cpf1 was introduced into S. elongatus UTEX 2973 as part of a CRISPRi system to target Photosystem I for inhibition [ 128 ]. Additionally, a plasmid-based CRISPRi system was introduced into S6803 [ 129 ] to inhibit the expression of the D1 protein of photosystem II, and Liu et al [ 130 ] introduced a reversible CRISPRi system into S6803 by using a Rhamnose-theophylline inducible promoter system. This CRISPRi system was able to almost completely knock out PSII by targeting the D2 ( psbD gene) protein, and it was fully reversible.…”

Section: Use Of Crispr and Crispr Interference For Study Of Photosynt...mentioning

confidence: 99%

Advances in the Understanding of the Lifecycle of Photosystem II

Johnson

Pakrasi

2022

Microorganisms

View full text Add to dashboard Cite

Photosystem II is a light-driven water-plastoquinone oxidoreductase present in cyanobacteria, algae and plants. It produces molecular oxygen and protons to drive ATP synthesis, fueling life on Earth. As a multi-subunit membrane-protein-pigment complex, Photosystem II undergoes a dynamic cycle of synthesis, damage, and repair known as the Photosystem II lifecycle, to maintain a high level of photosynthetic activity at the cellular level. Cyanobacteria, oxygenic photosynthetic bacteria, are frequently used as model organisms to study oxygenic photosynthetic processes due to their ease of growth and genetic manipulation. The cyanobacterial PSII structure and function have been well-characterized, but its lifecycle is under active investigation. In this review, advances in studying the lifecycle of Photosystem II in cyanobacteria will be discussed, with a particular emphasis on new structural findings enabled by cryo-electron microscopy. These structural findings complement a rich and growing body of biochemical and molecular biology research into Photosystem II assembly and repair.

show abstract

“…Escherichia coli strain DH5α was used for routine DNA manipulations [ 21 ] and constructions in Luria broth (LB) medium at 37°C [ 22 ]…”

Section: Methodsmentioning

confidence: 99%

Increased sensitivity of heavy metal bioreporters in transporter deficient Synechocystis PCC6803 mutants

et al. 2021

Self Cite

View full text Add to dashboard Cite

The detection and identification of heavy metal contaminants are becoming increasingly important as environmental pollution causes an ever-increasing health hazard in the last decades. Bacterial heavy metal reporters, which constitute an environmentally friendly and cheap approach, offer great help in this process. Although their application has great potential in the detection of heavy metal contamination, their sensitivity still needs to be improved. In this study, we describe a simple molecular biology approach to improve the sensitivity of bacterial heavy metal biosensors. The constructs are luxAB marker genes regulated by the promoters of heavy metal exporter genes. We constructed a mutant strain lacking the cluster of genes responsible for heavy metal transport and hence achieved increased intracellular heavy metal content of the Synechocystis PCC6803 cyanobacterium. Taking advantage of this increased intracellular heavy metal concentration the Ni2+; Co2+ and Zn2+ detection limits of the constructs were three to tenfold decreased compared to the sensitivity of the same constructs in the wild-type cyanobacterium.

show abstract

A single plasmid based CRISPR interference in Synechocystis 6803 – A proof of concept

Cited by 18 publications

References 37 publications

CRISPRi as a Tool to Repress Multiple Copies of Extracellular Polymeric Substances (EPS)-Related Genes in the Cyanobacterium Synechocystis sp. PCC 6803

CRISPRi as a Tool to Repress Multiple Copies of Extracellular Polymeric Substances (EPS)-Related Genes in the Cyanobacterium Synechocystis sp. PCC 6803

Advances in the Understanding of the Lifecycle of Photosystem II

Increased sensitivity of heavy metal bioreporters in transporter deficient Synechocystis PCC6803 mutants

Contact Info

Product

Resources

About