CRISPR–Cas9 System for Genome Engineering of Photosynthetic Microalgae

Patel, Vikas; Soni, Niraja; Prasad, Vandana; Sapre, Ajit; Dasgupta, Santanu; Bhadra, Bhaskar

doi:10.1007/s12033-019-00185-3

Cited by 56 publications

(35 citation statements)

References 104 publications

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“…These problems can be overcome by the recently developed gene-editing techniques. Gene-editing techniques based on RNP using Cas9 proteins are being recognized as the most effective gene specific knockout methods to date (Patel et al, 2019). Cas9-mediated gene knock-out has been reported for several genes and the use of donor DNA with RNP, called knock-in, has emerged recently.…”

Section: Discussionmentioning

confidence: 99%

Site-Specific Gene Knock-Out and On-Site Heterologous Gene Overexpression in Chlamydomonas reinhardtii via a CRISPR-Cas9-Mediated Knock-in Method

et al. 2020

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Chlamydomonas reinhardtii is being transformed from a model organism to an industrial organism for the production of pigments, fatty acids, and pharmaceuticals. Genetic modification has been used to increase the economic value of C. reinhardtii. However, low gene-editing efficiency and position-effects hinder the genetic improvement of this microorganism. Recently, site-specific double-stranded DNA cleavage using CRISPR-Cas9 system has been applied to regulate a metabolic pathway in C. reinhardtii. In this study, we proved that site-specific gene expression can be induced by CRISPR-Cas9mediated double-strand cleavage and non-homologous end joining (NHEJ) mechanism. The CRISPR-Cas9-mediated knock-in method was adopted to improve gene-editing efficiency and express the reporter gene on the intended site. Knock-in was performed using a combination of ribonucleoprotein (RNP) complex and DNA fragment (antibiotics resistance gene). Gene-editing efficiency was improved via optimization of a component of RNP complex. We found that when the gene CrFTSY was targeted, the efficiency of obtaining the desired mutant by the knock-in method combined with antibiotic resistance was nearly 37%; 2.5 times higher than the previous reports. Additionally, insertion of a long DNA fragment (3.2 and 6.4 kb) and site-specific gene expression were analyzed. We demonstrated the knockout phenotype of CrFTSY and on-site inserted gene expression of luciferase and mVenus at the same time. This result showed that CRISPR-Cas9-mediated knock-in can be used to express the gene of interest avoiding position-effects in C. reinhardtii. This report could provide a new perspective to the use of gene-editing. Furthermore, the technical improvements in genetic modification may accelerate the commercialization of C. reinhardtii.

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Section: Discussionmentioning

confidence: 99%

Site-Specific Gene Knock-Out and On-Site Heterologous Gene Overexpression in Chlamydomonas reinhardtii via a CRISPR-Cas9-Mediated Knock-in Method

et al. 2020

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“…The ability to change, remove or introduce gene(s) of interest in a programmable way, and to achieve a stable phenotype of the engineered strains over time, is pivotal to industrial application of photosynthetic bio-factories (Patel et al, 2019;Pérez et al, 2019). Molecular tools are better established for cyanobacteria than for eukaryotic algae, but nuclease-based genome editing tools like Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and Transcription Activator-Like Effector Nucleases (TALEN) are rapidly developing and applied to algal species of different phylogeny and commercial value (Doron et al, 2016;Nymark et al, 2016;Shin et al, 2016;Kroth et al, 2018;Verruto et al, 2018;Ortega-Escalante et al, 2019;Pérez et al, 2019;Ng et al, 2020).…”

Section: Molecular Biology Tools: State-of-the-art and Open Challengesmentioning

confidence: 99%

“…Such weaknesses have been mainly ascribed to positional effects of gene integration and/or gene silencing. Secondary off-target mutations in edited strains, even when targeted genome editing has been performed with nucleases (Patel et al, 2019), are an undesirable aspect of algae transformation. To minimize issues due to offtarget mutations, ribonucleoproteins (RNP-) based methods for CRISPR/Cas gene editing have been developed in substitution to vector-mediated protocols (Patel et al, 2019).…”

Section: Molecular Biology Tools: State-of-the-art and Open Challengesmentioning

confidence: 99%

Toward Enhanced Fixation of CO2 in Aquatic Biomass: Focus on Microalgae

2020

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The need to reduce the CO 2 footprint of human activities calls for the utilization of new means of production and new sources of products. Microalgae are a very promising source of a large variety of products, from fuels to chemicals for multiple industrial applications (e.g., dyes, pharmaceutical products, cosmetics, food and feed, new materials for high tech manufacture), and for processes such as wastewater treatment. Algae, as photosynthetic organisms, use light to energize the synthesis of organic matter and differently from most terrestrial plants, can be cultured on land that is not used for crop production. We describe the main factors contributing to microalgae productivity in artificial cultivation systems and discuss the research areas that still need investigation in order to pave the way to the generation of photosynthetic cell factories. We shall comment on the main caveats of the possible mode of improving photosynthetic efficiency and to optimize the partitioning of fixed C to products of commercial relevance. We address the problem of the selection of the appropriate strain and of the consequences of their diverse physiology and culture conditions for a successful commercial application. Finally, we shall provide state of the art information on cell factories chassis by means of synthetic biology approaches to produce chemicals of interest.

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“…Although genetic engineering of algae is still in the initial stages, the use of this technique to enhance lutein content in microalgae has made progress in recent years. For example, new molecular technologies such as CRISPR/Cas9 genome engineering technology and genome sequencing have improved understanding of lutein metabolism regulation in algae species [ 34 , 35 , 36 , 37 ].…”

Section: Introductionmentioning

confidence: 99%

Challenges and Potential in Increasing Lutein Content in Microalgae

2021

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Research on enhancing lutein content in microalgae has made significant progress in recent years. However, strategies are needed to address the possible limitations of microalgae as practical lutein producers. The capacity of lutein sequestration may determine the upper limit of cellular lutein content. The preliminary estimation presented in this work suggests that the lutein sequestration capacity of the light-harvesting complex (LHC) of microalgae is most likely below 2% on the basis of dry cell weight (DCW). Due to its nature as a structural pigment, higher lutein content might interfere with the LHC in fulfilling photosynthetic functions. Storing lutein in a lipophilic environment is a mechanism for achieving high lutein content but several critical barriers must be overcome such as lutein degradation and access to lipid droplet to be stored through esterification. Understanding the mechanisms underlying lipid droplet biogenesis in chloroplasts, as well as carotenoid trafficking through chloroplast membranes and carotenoid esterification, may provide insight for new approaches to achieve high lutein contents in algae. In the meantime, building the machinery for esterification and sequestration of lutein and other hydroxyl-carotenoids in model microorganisms, such as yeast, with synthetic biology technology provides a promising option.

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CRISPR–Cas9 System for Genome Engineering of Photosynthetic Microalgae

Cited by 56 publications

References 104 publications

Site-Specific Gene Knock-Out and On-Site Heterologous Gene Overexpression in Chlamydomonas reinhardtii via a CRISPR-Cas9-Mediated Knock-in Method

Site-Specific Gene Knock-Out and On-Site Heterologous Gene Overexpression in Chlamydomonas reinhardtii via a CRISPR-Cas9-Mediated Knock-in Method

Toward Enhanced Fixation of CO2 in Aquatic Biomass: Focus on Microalgae

Challenges and Potential in Increasing Lutein Content in Microalgae

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