Precision genetic modifications: a new era in molecular biology and crop improvement

Fichtner, Franziska; Castellanos, Reynel Urrea; Ülker, Bekir

doi:10.1007/s00425-014-2029-y

Cited by 50 publications

(36 citation statements)

References 161 publications

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“…The first set of plasmids contain Golden Gate entry clones, each carrying a complete expression cassette for one gRNA under either the Arabidopsis U6 (AtU6) or AtU3 promoter (for dicot plants) or the rice (Oryza sativa) U6 (OsU6) or OsU3 promoter (for monocot plants; Fig. 2A; Table I; Fichtner et al, 2014). The first step is to clone individual gRNAs into these Golden Gate entry clones by a simple digestion (with Type IIS enzyme Esp3I or BsmBI) and ligation step.…”

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confidence: 99%

A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation

et al. 2015

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The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied plant research.

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confidence: 99%

A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation

et al. 2015

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“…We can also alter gene expression by using a dead or broken Cas9 enzyme to block the binding of RNA polymerase needed for the gene to be expressed or attaching a dead or broken Cas9 protein to transcriptional factors or activator to stimulate the gene expression [29,30]. Furthermore, CRISPRCas9 was used to construct somatic and germline mouse models with point mutations or chromosomal deletions in multiple genes using multiple gRNAs, and even more complex chromosomal rearrangements.…”

Section: Recent Advances and Applicationsmentioning

confidence: 99%

CRISPR/Cas9-Mediated Genome Nano-Surgery: An Update

Mokbel¹,

Mokbel²

2017

Biochem Mol biol J

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Recent advances in sequencing methods have prompted an upsurge in research into the modification of diseaseassociated genes or genes involved in drug resistance. In the early days of genome-editing, immunogenic and difficult to deliver tools with high off-target effects such as Zinc Finger Nucleases (ZFNs) and Transcription activatorlike effector nucleases (TALENs) proteins were used. This was followed by the discovery of the Clustered Regularly Interspaced Short Palindromic Repeat s, CRISPR/Cas9 system. This "self-non-self-discriminatory" natural defence mechanism in bacteria and archaea identifies and degrades extrachromosomal genomes or foreign genetic elements to prevent them from integrating into the prokaryotic genomes. Unlike traditional gene therapies that can merely insert a gene in a random pattern, the inexpensive and expedient CRISPR/ Cas9 system was harnessed by scientists to precisely cut, add or manipulate multiple DNA sequences at specific sites. This review will focus on the most recent studies in genome-editing by giving an overview of the past and modern methods in this field with an emphasis on the bacterial adaptive immune system called CRISPR/Cas9. This article will also analyze the applications of CRISPR/ Cas9 in rewriting the human genome in clinical and research settings and its potential future therapeutic applications. In addition, we shall consider the technical complexities which need to be overcome for the safe and effective delivery of this novel therapy and how the ethical concerns associated with this revolutionary genome engineering technique have constrained research in germ-line genome-editing. Furthermore, we shall highlight the role of scientific regulatory committees in evaluating and assessing safety issues such as potential complications before translating this proof-of-concept evidential approach to clinical reality. More research is required to explore the risk of hazardous genome edits and to fine-tune and improve this novel therapeutic system. Although the inexpensive and easy to use CRISPR/ Cas9 platform paves new ways for precise genome editing, clinical application is still at an early stage.

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“…In all cases, a continuing issue is the delivery of all the reagents efficiently and functionally to the cells or organisms under study. The CRISPR/CRISPR-associated protein 9 (Cas9) tool seems to overcome some of the shortcomings of the other methods [76,77]. Successful examples of targetable nucleases application are reported for Arabidopsis, tobacco, rice, maize, soybean, barley, cabbage, and bunchgrass by using different delivery technologies, including T-DNA plasmid from Agrobacterium, protoplasts and embryonic callus manipulation, and subsequent plant regeneration [70,[78][79][80][81][82].…”

Section: Genome Editingmentioning

confidence: 99%

Perspectives on the Application of Next-generation Sequencing to the Improvement of Africa’s Staple Food Crops

Gedil¹,

Ferguson²,

Girma³

et al. 2016

Next Generation Sequencing - Advances, Applications and Challenges

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The persistent challenge of insufficient food, unbalanced nutrition, and deteriorating natural resources in the most vulnerable nations, characterized by fast population growth, calls for utilization of innovative technologies to curb constraints of crop production. Enhancing genetic gain by using a multipronged approach that combines conventional and genomic technologies for the development of stress-tolerant varieties with high yield and nutritional quality is necessary. The advent of nextgeneration sequencing (NGS) technologies holds the potential to dramatically impact the crop improvement process. NGS enables whole-genome sequencing (WGS) and re-sequencing, transcriptome sequencing, metagenomics, as well as high-throughput genotyping, which can be applied for genome selection (GS). It can also be applied to diversity analysis, genetic and epigenetic characterization of germplasm and pathogen detection, identification, and elimination. High-throughput phenotyping, integrated data management, and decision support tools form the necessary supporting environment for effective utilization of genome sequence information. It is important that these opportunities for mainstreaming innovative breeding strategies, enabled by cutting-edge "Omics" technologies, are seized in Africa; however, several constraints must be addressed before the benefit of NGS can be fully realized. African breeding programs must have access to high-throughput genotyping facilities, capacity in the application of genome selection and marker-assisted breeding must be built and supported by capacity in genomic analysis and bioinformatics. This chapter demonstrates how interventions with NGS-enabled innovative strategies can be applied to increase genetic gain with insights from the Consortium of International

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Precision genetic modifications: a new era in molecular biology and crop improvement

Cited by 50 publications

References 161 publications

A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation

A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation

CRISPR/Cas9-Mediated Genome Nano-Surgery: An Update

Perspectives on the Application of Next-generation Sequencing to the Improvement of Africa’s Staple Food Crops

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