Progress of CRISPR‐Cas Based Genome Editing in Photosynthetic Microbes

Naduthodi, Mihris Ibnu Saleem; Barbosa, María J.; Oost, John van der

doi:10.1002/biot.201700591

Cited by 45 publications

(42 citation statements)

References 55 publications

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“…Recent studies toward the photosynthetic production of useful chemicals have rapidly developed tools for artificial gene regulation systems for cyanobacteria (25)(26)(27). Among them, a gene knockdown technology, CRISPR interference (CRISPRi), has attracted much attention because this system exhibited repression over a wide dynamic range in an inducer concentration-dependent manner (28)(29)(30)(31)(32). Target genes can be repressed by the formation of a complex consisting of a nuclease-deficient Cas9 (dCas9); a single guide RNA (sgRNA), which corresponds to the target DNA sequence; and target DNA (33).…”

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confidence: 99%

cyAbrB Transcriptional Regulators as Safety Devices To Inhibit Heterocyst Differentiation in Anabaena sp. Strain PCC 7120

et al. 2019

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Cyanobacteria are monophyletic organisms that perform oxygenic photosynthesis. While they exhibit great diversity, they have a common set of genes. However, the essentiality of them for viability has hampered the elucidation of their functions. One example of these genes is cyabrB1 (also known as calA in Anabaena sp. strain PCC 7120), encoding a transcriptional regulator. In the present study, we investigated the function of calA/cyabrB1 in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 through CRISPR interference, a method that we recently utilized for the photosynthetic production of a useful chemical in this strain. Conditional knockdown of calA/cyabrB1 in the presence of nitrate resulted in the formation of heterocysts. Two genes, hetP and hepA, which are required for heterocyst formation, were upregulated by calA/cyabrB1 knockdown in the presence of combined nitrogen sources. These genes are known to be induced by HetR, a master regulator of heterocyst formation. hetR was not induced by calA/cyabrB1 knockdown. hetP and hepA were repressed by direct binding of CalA/cyAbrB1 to their promoter regions in a HetR-independent manner. In addition, the overexpression of calA/cyabrB1 abolished heterocyst formation upon nitrogen depletion. Also, knockout of calB/cyabrB2 (a paralogue gene of calA/cyabrB1), in addition to knockdown of calA/cyabrB1, enhanced heterocyst formation in the presence of nitrate, suggesting functional redundancy of cyAbrB proteins. We propose that a balance between amounts of HetR and CalA/cyAbrB1 is a key factor influencing heterocyst differentiation during nitrogen stepdown. We concluded that cyAbrB proteins are essential safety devices that inhibit heterocyst differentiation. IMPORTANCE Spore formation in Bacillus subtilis and Streptomyces has been extensively studied as models of prokaryotic nonterminal cell differentiation. In these organisms, many cells/hyphae differentiate simultaneously, which is governed by a network in which one regulator stands at the top. Differentiation of heterocysts in Anabaena sp. strain PCC 7120 is unique because it is terminal, and only 5 to 10% of vegetative cells differentiate into heterocysts. In this study, we identified CalA/cyAbrB1 as a repressor of two genes that are essential for heterocyst formation independently of HetR, a master activator for heterocyst differentiation. This finding is reasonable for unique cell differentiation of Anabaena because CalA/cyAbrB1 could suppress heterocyst differentiation tightly in vegetative cells, while only cells in which HetR is overexpressed could differentiate into heterocysts.

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cyAbrB Transcriptional Regulators as Safety Devices To Inhibit Heterocyst Differentiation in Anabaena sp. Strain PCC 7120

et al. 2019

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“…The higher copy number of exogenous plasmids per chromosome, as reported for RSF1010 and pANS derivatives, can increase the gene expression level. Importantly, the availability of stable and fine‐tuned plasmids will facilitate the utilization of the genetic toolkits by, for example, carrying “helper genes” (e.g., recombinases and nucleases) to perform “one‐time” tasks, such as recombination and DNA cleavage . Moreover, exogenous plasmids are more convenient for “proof‐of‐principle” studies because they eliminate the segregation process and make strain engineering less time‐consuming.…”

Section: Expression Of Heterologous Genesmentioning

confidence: 99%

Synthetic Biology Toolkits for Metabolic Engineering of Cyanobacteria

Xia

Ling

Foo

et al. 2019

Biotechnology Journal

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Cyanobacteria are of great importance to Earth's ecology. Due to their capability in photosynthesis and C1 metabolism, they are ideal microbial chassis that can be engineered for direct conversion of carbon dioxide and solar energy into biofuels and biochemicals. Facilitated by the elucidation of the basic biology of the photoautotrophic microbes and rapid advances in synthetic biology, genetic toolkits have been developed to enable implementation of nonnatural functionalities in engineered cyanobacteria. Hence, cyanobacteria are fast becoming an emerging platform in synthetic biology and metabolic engineering. Herein, the progress made in the synthetic biology toolkits for cyanobacteria and their utilization for transforming cyanobacteria into microbial cell factories for sustainable production of biofuels and biochemicals is outlined. Current techniques in heterologous gene expression, strategies in genome editing, and development of programmable regulatory parts and modules for engineering cyanobacteria towards biochemical production are discussed and prospected. As cyanobacteria synthetic biology is still in its infancy, apart from the achievements made, the difficulties and challenges in applying and developing genetic toolkits in cyanobacteria for biochemical production are also evaluated.

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“…However, modifications in the Cas9 protein delivery through the ribonucleoprotein (RNP) complex has been reported to reduce the off-target problems associated with the Cas9 protein. Replacing the Cas9 protein with a Cas12a variant is also an alternative to consider as it has been reported to solve the cytotoxicity in cyanobacteria (Naduthodi et al, 2018). Apart from these, the recent characterization of several other variants of the CRISPR system has extended the prospect of a genetic toolbox for microalgal genome engineering.…”

Section: Genome Editing In Microalgae For Strain Improvementmentioning

confidence: 99%

Tailoring Microalgae for Efficient Biofuel Production

et al. 2018

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Depleting fossil fuel, soaring prices, growing demand, and global climate change concerns have driven the research for finding an alternative source of sustainable fuel. Microalgae have emerged as a potential feedstock for biofuel production as many strains accumulate higher amounts of lipid, with faster biomass growth and higher photosynthetic yield than their land plant counterparts. In addition to this, microalgae can be cultured without needing agricultural land or ecological landscapes and offer opportunities for mitigating global climate change, allowing waste water treatment and carbon dioxide sequestration. Despite these benefits, microalgae pose many challenges, including low lipid yield under limiting growth conditions and slow growth in high lipid content strains. Biotechnological interventions can make major advances in strain improvement for the commercial scale production of biofuel. We discuss various strategies, including efficient transformation toolbox, to increase lipid accumulation and its quality through the regulation of key enzymes involved in lipid production, by blocking the competing pathways, pyramiding genes, enabling high cell biomass under nutrient-deprived conditions and other environmental stresses, and controlling the upstream regulators of targets, the transcription factors, and microRNAs. We highlight the opportunities emerging from the current progress in the application of genome editing in microalgae for accelerating the strain improvement program.

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Progress of CRISPR‐Cas Based Genome Editing in Photosynthetic Microbes

Cited by 45 publications

References 55 publications

cyAbrB Transcriptional Regulators as Safety Devices To Inhibit Heterocyst Differentiation in Anabaena sp. Strain PCC 7120

cyAbrB Transcriptional Regulators as Safety Devices To Inhibit Heterocyst Differentiation in Anabaena sp. Strain PCC 7120

Synthetic Biology Toolkits for Metabolic Engineering of Cyanobacteria

Tailoring Microalgae for Efficient Biofuel Production

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