Highly efficient transgene‐free targeted mutagenesis and single‐stranded oligodeoxynucleotide‐mediated precise knock‐in in the industrial microalga <i>Euglena gracilis</i> using Cas9 ribonucleoproteins

Nomura, Toshihisa; Inoue, Komaki; Uehara‐Yamaguchi, Yukiko; Yamada, Koji; Iwata, Osamu; Suzuki, Kengo; Mochida, Keiichi

doi:10.1111/pbi.13174

Cited by 30 publications

(34 citation statements)

References 10 publications

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“…The applications of the CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR associated proteins) 9 system in this field are also pertinent since they could allow suppressing competitive pathways; increasing the production of specific molecules [ 207 , 208 ]. In this sense; RNA interference (RNAi) is another important tool to address this suppression.…”

Section: Perspectivesmentioning

confidence: 99%

The Potential of Algal Biotechnology to Produce Antiviral Compounds and Biopharmaceuticals

et al. 2020

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The emergence of the Coronavirus Disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to an unprecedented pandemic, which demands urgent development of antiviral drugs and antibodies; as well as prophylactic approaches, namely vaccines. Algae biotechnology has much to offer in this scenario given the diversity of such organisms, which are a valuable source of antiviral and anti-inflammatory compounds that can also be used to produce vaccines and antibodies. Antivirals with possible activity against SARS-CoV-2 are summarized, based on previously reported activity against Coronaviruses or other enveloped or respiratory viruses. Moreover, the potential of algae-derived anti-inflammatory compounds to treat severe cases of COVID-19 is contemplated. The scenario of producing biopharmaceuticals in recombinant algae is presented and the cases of algae-made vaccines targeting viral diseases is highlighted as valuable references for the development of anti-SARS-CoV-2 vaccines. Successful cases in the production of functional antibodies are described. Perspectives on how specific algae species and genetic engineering techniques can be applied for the production of anti-viral compounds antibodies and vaccines against SARS-CoV-2 are provided.

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Section: Perspectivesmentioning

confidence: 99%

The Potential of Algal Biotechnology to Produce Antiviral Compounds and Biopharmaceuticals

et al. 2020

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“…Strains created using the genome editing technology employing the ribonucleoprotein (RNP) complex are transgene-free and are suitable for industrial use. At present, RNP-based genome editing of microalgae using CRISPR-Cas9 ( Cong and Zhang, 2014 ) has been actively performed in various species, but many selection-free methods display a low mutation efficiency (∼1%) ( Table 2 ; Baek et al, 2016 ; Shin et al, 2016 ; Ferenczi et al, 2017 ; Serif et al, 2018 ; Yoshimitsu et al, 2018 ; Findinier et al, 2019 ; Naduthodi et al, 2019 ; Nomura et al, 2019 ). In E. gracilis , a highly efficient genome editing method was recently established based on a Cas9-RNP introduction procedure, which does not depend on the expression of the transgene in the cell ( Figure 2 ).…”

Section: Research Challenges Involved In the Genetic Modification Of mentioning

confidence: 99%

“…In the first case, the Cas9 RNP targeting the gene EgGSL2 involved in the biosynthesis of paramylon in E. gracilis ( Tanaka et al, 2017 ) was introduced using electroporation. Direct delivery of Cas9 RNPs into the cell achieved a non-homologous end joining-mediated indel mutation rate of up to 90.1% ( Nomura et al, 2019 ). This rate is much higher than the mutation efficiency of Chlamydomonas (∼10%) and other microalgae (∼1%) when using the RNP-based genome editing method ( Jeon et al, 2017 ; Spicer and Molnar, 2018 ).…”

Section: Research Challenges Involved In the Genetic Modification Of mentioning

confidence: 99%

“…This rate is much higher than the mutation efficiency of Chlamydomonas (∼10%) and other microalgae (∼1%) when using the RNP-based genome editing method ( Jeon et al, 2017 ; Spicer and Molnar, 2018 ). In addition, using single-stranded oligodeoxynucleotide as donor DNA template, precise knock-in (∼70%) of 42 bp exogenous DNA sequences via homology-induced repair has been successfully achieved ( Nomura et al, 2019 ). In many microalgae, except E. gracilis , the lower efficiency of genome editing is related to the presence of cell walls or structures on the cell surface ( Jeon et al, 2017 ).…”

Section: Research Challenges Involved In the Genetic Modification Of mentioning

confidence: 99%

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Genetic Engineering Strategies for Euglena gracilis and Its Industrial Contribution to Sustainable Development Goals: A Review

Harada¹,

Nomura

Yamada³

et al. 2020

Front. Bioeng. Biotechnol.

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The sustainable development goals (SDGs) adopted at the 2015 United Nations Summit are globally applicable goals designed to help countries realize a sustainable future. To achieve these SDGs, it is necessary to utilize renewable biological resources. In recent years, bioeconomy has been an attractive concept for achieving the SDGs. Microalgae are one of the biological resources that show promise in realizing the “5F”s (food, fiber, feed, fertilizer, and fuel). Among the microalgae, Euglena gracilis has the potential for achieving the “5F”s strategy owing to its unique features, such as production of paramylon, that are lacking in other microalgae. E. gracilis has already been produced on an industrial scale for use as an ingredient in functional foods and cosmetics. In recent years, genetic engineering methods for breeding E. gracilis have been researched and developed to achieve higher yields. In this article, we summarize how microalgae contribute toward achieving the SDGs. We focus on the contribution of E. gracilis to the bioeconomy, including its advantages in industrial use as well as its unique characteristics. In addition, we review genetic engineering-related research trends centered on E. gracilis , including a complete nuclear genome determination project, genome editing technology using the CRISPR-Cas9 system, and the development of a screening method for selecting useful strains. In particular, genome editing in E. gracilis could be a breakthrough for molecular breeding of industrially useful strains because of its high efficiency.

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“…Therefore, this method is suitable for basic research and industrial applications, such as the breeding of Euglena . For complete details on the use and execution of this protocol, please refer to Nomura et al. (2019) .…”

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confidence: 99%

Highly Efficient CRISPR-Associated Protein 9 Ribonucleoprotein-Based Genome Editing in Euglena gracilis

Nomura

Yoshikawa²,

Suzuki³

et al. 2020

STAR Protocols

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Summary Euglena gracilis , a unicellular phytoflagellate microalga, is a promising biomaterial for foods, feeds, and biofuels. However, targeted mutagenesis in this species has been a long-standing challenge. We recently developed a transgene-free, highly efficient, genome editing method for E. gracilis using CRISPR/Cas9 ribonucleoproteins (RNPs). Our method achieved mutagenesis rates of approximately 80% or more through an electroporation-based direct delivery of Cas9 RNPs. Therefore, this method is suitable for basic research and industrial applications, such as the breeding of Euglena . For complete details on the use and execution of this protocol, please refer to Nomura et al. (2019) .

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Highly efficient transgene‐free targeted mutagenesis and single‐stranded oligodeoxynucleotide‐mediated precise knock‐in in the industrial microalga Euglena gracilis using Cas9 ribonucleoproteins

Cited by 30 publications

References 10 publications

The Potential of Algal Biotechnology to Produce Antiviral Compounds and Biopharmaceuticals

The Potential of Algal Biotechnology to Produce Antiviral Compounds and Biopharmaceuticals

Genetic Engineering Strategies for Euglena gracilis and Its Industrial Contribution to Sustainable Development Goals: A Review

Highly Efficient CRISPR-Associated Protein 9 Ribonucleoprotein-Based Genome Editing in Euglena gracilis

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