Abstract
The carbon footprint caused by unsustainable development and its environmental and economic impact has become a major concern in the past few decades. Photosynthetic microbes such as microalgae and cyanobacteria are capable of accumulating value-added compounds from carbon dioxide, and have been regarded as environmentally friendly alternatives to reduce the usage of fossil fuels, thereby contributing to reducing the carbon footprint. This light-driven generation of green chemicals and biofuels has triggered the research for metabolic engineering of these photosynthetic microbes. CRISPR-Cas systems are successfully implemented across a wide range of prokaryotic and eukaryotic species for efficient genome editing. However, the inception of this genome editing tool in microalgal and cyanobacterial species took off rather slowly due to various complications. In this review, we elaborate on the established CRISPR-Cas based genome editing in various microalgal and cyanobacterial species. The complications associated with CRISPR-Cas based genome editing in these species are addressed along with possible strategies to overcome these issues. It is anticipated that in the near future this will result in improving and expanding the microalgal and cyanobacterial genome engineering toolbox.
Highlights
The carbon footprint caused by unsustainable development and its environworld
In line with the observation made in other organisms, the Homology Directed Repair (HDR) was found to be enhanced in PCC 7942 by the induction of DSB by Cas9.[6,18,45] This Cas9-assisted HDR was used for metabolic engineering of PCC 7942 by knocking in genes coding for phosphoenolpyruvate carboxylase and citrate synthase to enhance the carbon flux towards oxidative pathway of TCA cycle
Genome editing in a wide variety of species has been simplified with efficient and successful application of the CRISPR-Cas technology
Summary
The exploitation of non-renewable energy sources to meet the perpetual requirement of increasing human population has resulted in their rapid depletion and a steady rise in price. Cas12a (Cpf1) of the type V system is gaining global attention for genome editing in various species.[10,11,12] Cas12a is an interesting alternative tool for genome engineering as it has distinct features compared to Cas (Figure 1), such as (i) Cas12a uses a single crRNA instead of a set of crRNA and trans-activating crRNA (tracrRNA) in Cas that is synthetically fused as a single guide RNA (sgRNA) that is at least twice as long as the crRNA guide used by Cas12a, (ii) Cas12a has been demonstrated to catalyze the maturation of its own crRNA which allows for efficient multiplex genome editing,[12,13] while maturation of crRNA:tracrRNA complex of Cas relies on processing by the non-Cas ribonuclease RNase III, (iii) Cas12a uses a T-rich PAM (50-TTTN-30) upstream the protospacer in contrast to the downstream located G-rich PAM (50-NGG-30) that is recognized by Cas9,[11] (iv) Cas12a generates staggered ends with five nucleotide overhangs in the target DNA, compared to the blunt end cleavage by Cas9,[11,14] (v) Cas12a has a single nuclease domain (RuvC) that cleaves 18–23 base pairs downstream from the PAM-proximal seed sequence, whereas cleavage by the two nuclease domains of Cas (RuvC and HNH) occurs within the seed three base pairs upstream its PAM (Figure 1). This review elaborates on the application of CRISPR-Cas based genome engineering in microalgae and cyanobacteria, and provides insights on strategies to enhance efficiency of the CRISPR-Cas tools for generating targeted mutants in these microbes (Table 1)
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