CRISPR/Cas9-mediated gfp gene inactivation in Arabidopsis suspension cells

Permyakova, N. V.; Sidorchuk, Yu. V.; Маренкова, Т. В.; Khozeeva, Sofya A; Кузнецов, В.В.; Zagorskaya, A. A.; Розов, С. М.; Дейнеко, Е. В.

doi:10.1007/s11033-019-05007-y

Cited by 12 publications

(6 citation statements)

References 45 publications

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“…Our GFP activation assay requires a cell sorting step to enrich the cleavage events before targeted amplicon sequencing, which increases the sensitivity. GFP-disruption assay can be used for detection of DNA cleavage ( Permyakova et al, 2019 ), but it cannot detect the rare cleavage events due to the background of GFP-negative cells. In addition, GFP-disruption assay does not allow us to test non-GFP DNA sequence.…”

Section: Discussionmentioning

confidence: 99%

A Highly Sensitive GFP Activation Assay for Detection of DNA Cleavage in Cells

Zhang

Wang

et al. 2021

Front. Cell Dev. Biol.

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CRISPR/Cas9 nucleases hold great potential for gene therapy, but they frequently induce unwanted off-target cleavage. We previously developed a GFP activation assay for detection of DNA cleavage in cells. Here, we demonstrate two novel applications of this assay. First, we use this assay to confirm off-target cleavage that cannot be detected by targeted deep sequencing in cells before. Second, we use this approach to detect multiple alternative PAMs recognized by SpCas9. These noncanonical PAMs are associated with low cleavage activity, but targets associated with these PAMs must be considered as potential off-target sites. Taken together, the GFP activation assay is a powerful platform for DNA cleavage detection in cells.

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Section: Discussionmentioning

confidence: 99%

A Highly Sensitive GFP Activation Assay for Detection of DNA Cleavage in Cells

Zhang

Wang

et al. 2021

Front. Cell Dev. Biol.

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“…Combined treatment of the mannosidase inhibitors kifunensine (KIF) and swainsonine (SWA) in transgenic rice cell culture media can be an effective method of producing recombinant human acid α-glucosidase (rhGAA) displaying dominantly high-mannose glycans such as Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 (Man7/8/9) glycoforms without genetic manipulation of glycosylation (Choi et al, 2018). In a recent study, knockout of a green fluorescent protein (gfp) reporter gene in Arabidopsis cell culture was carried out, and it was concluded that the CRISPR/Cas9 system can be utilized for introducing site-specific mutations into the genome of cultured suspension cells of Arabidopsis (Permyakova et al, 2019). A new plant system based on carnivorous plants was established and showed the ability of biomimetic approaches to lead to an original production of recombinant proteins.…”

Section: Transgenic Plantsmentioning

confidence: 99%

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

Tripathi

Shrivastava

2019

Front. Bioeng. Biotechnol.

369

243

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Infectious diseases, along with cancers, are among the main causes of death among humans worldwide. The production of therapeutic proteins for treating diseases at large scale for millions of individuals is one of the essential needs of mankind. Recent progress in the area of recombinant DNA technologies has paved the way to producing recombinant proteins that can be used as therapeutics, vaccines, and diagnostic reagents. Recombinant proteins for these applications are mainly produced using prokaryotic and eukaryotic expression host systems such as mammalian cells, bacteria, yeast, insect cells, and transgenic plants at laboratory scale as well as in large-scale settings. The development of efficient bioprocessing strategies is crucial for industrial production of recombinant proteins of therapeutic and prophylactic importance. Recently, advances have been made in the various areas of bioprocessing and are being utilized to develop effective processes for producing recombinant proteins. These include the use of high-throughput devices for effective bioprocess optimization and of disposable systems, continuous upstream processing, continuous chromatography, integrated continuous bioprocessing, Quality by Design, and process analytical technologies to achieve quality product with higher yield. This review summarizes recent developments in the bioprocessing of recombinant proteins, including in various expression systems, bioprocess development, and the upstream and downstream processing of recombinant proteins.

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“…Plant cells suspension cultures are the valuable and renewable resource of biological material that can be used for several applications, including production of potential secondary metabolites (Yue et al 2016;Salehi et al 2018;Santos et al 2019). The plant cells suspension cultures are gaining popularity as a host system for the production of recombinant proteins (Tekoah et al 2015;Yue et al 2016) and have several advantages such as post-translational modi cations, a slight risk of viral contamination, low cost of plant culture media, and cost-e ciency of bacterial expression systems (Santos et al 2016;Zagorskaya and Deineko 2017;Permyakova et al 2019). Metabolic engineering requires a deep understanding of its molecular and genetic architecture to produce targeted secondary metabolites successfully.…”

Section: Introductionmentioning

confidence: 99%

“…The suspension cells can be developed from any explant type; however, most preferably, an embryogenic callus is used. Several successful cell suspension cultures were reported from various plants such as Artemisia annua (Salehi et al 2019), Catharanthus (Saiman et al 2018), Taxus (Wilson et al 2018), Capsicum (Brito-Sanchez et al 2019, Medicago (Santos et al 2019), Arabidopsis (Permyakova et al 2019), Gentiana (Rybczynski andWojcik 2019), andPanicum (Ondzighi-Assoume et al 2019). Agrobacterium-mediated genetic transformation depends on factors like types of explants, bacterial density, co-cultivation duration, the temperature of co-cultivation, concentration of acetosyringone, etc.…”

Section: Introductionmentioning

confidence: 99%

“…Hence, it is necessary to develop metabolic engineering methods to optimize the production for different plant systems (Wilson et al, 2014). A reliable and stable gene transfer method must be designed for metabolic engineering of cell suspension cultures (Wilson et al, 2018), which could enable the successful utilization of suspension cultures for a wide range of studies such as molecular biology, biochemistry, and genome editing (Permyakova et al, 2019). Various factors like optical density (OD) of bacterial culture, co-cultivation time, co-cultivation temperature, silwet, pluronic concentration, acetosyringone, etc., were evaluated to develop a reliable and high-throughput stable transformation method.…”

Section: Introductionmentioning

confidence: 99%

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Agrobacterium-Mediated Genetic Transformation and Cloning of Reference Genes in Suspension Cells of Artemisia Pallens

Jogam

Sandhya

Alok

et al. 2021

Preprint

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A reliable and stable Agrobacterium-mediated genetic transformation system has been developed using cell suspension cultures derived from Artemisia pallens cotyledon explants. Cotyledon, attached cotyledon, and compound leaf were found to be suitable for the induction of callus among five different types of explants tested. Yellow friable callus derived from attached cotyledon was used to initiate suspension cultures in Suspension Culture Medium (SCM) which was supplemented with 2.4-dichlorophenoxyacetic acid (2,4-D) and in combination with different concentrations of Zeatin (ZEA). Among the two different shock treatments, cold shock (at 4 oC) for 20 minutes and heat shock (at 45oC) treatment for 5 minutes, heat shock treatment increased the transformation efficiency. Supplementation of chemical additives such as Silwet L-77 (0.05%) and Pluronic F-68 (0.05%) significantly enhanced suspension cultures' transformation efficiency. The maximum GUS intensity was recorded with an optimal intensity of blue spots in the transformed cells. The highest GUS fluorometric activity was measured as 879.4±113.7 nmol 4MU/mg/min in transformed cell suspension cultures. The hygromycin-resistant callus derived from micro-calli showed intense blue colour in GUS histochemical assay. The transgene integration into the plant genome was confirmed by polymerase chain reaction (PCR) using uidA specific primers in six hygromycin-resistant cell lines. The cloned and mRNA expression levels of three candidate reference genes ADP-ribosylation factor (Arf), β-actin (Act), and ubiquitin (Ubi), and carotenoid biosynthesis pathway gene, i.e., Phytoene desaturase (Pds) along with transgene (uidA) were evaluated in transgenic callus lines. The present Agrobacterium-mediated genetic transformation protocol could help in better understand the metabolic pathways of this medicinally important plant and its genetic improvement.

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CRISPR/Cas9-mediated gfp gene inactivation in Arabidopsis suspension cells

Cited by 12 publications

References 45 publications

A Highly Sensitive GFP Activation Assay for Detection of DNA Cleavage in Cells

A Highly Sensitive GFP Activation Assay for Detection of DNA Cleavage in Cells

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

Agrobacterium-Mediated Genetic Transformation and Cloning of Reference Genes in Suspension Cells of Artemisia Pallens

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