The Clustered Regularly Interspaced Short Palindromic Repeats associated Cas9/sgRNA system is a novel targeted genome-editing technique derived from bacterial immune system. It is an inexpensive, ...easy, most user friendly and rapidly adopted genome editing tool transforming to revolutionary paradigm. This technique enables precise genomic modifications in many different organisms and tissues. Cas9 protein is an RNA guided endonuclease utilized for creating targeted double-stranded breaks with only a short RNA sequence to confer recognition of the target in animals and plants. Development of genetically edited (GE) crops similar to those developed by conventional or mutation breeding using this potential technique makes it a promising and extremely versatile tool for providing sustainable productive agriculture for better feeding of rapidly growing population in a changing climate. The emerging areas of research for the genome editing in plants include interrogating gene function, rewiring the regulatory signaling networks and sgRNA library for high-throughput loss-of-function screening. In this review, we have described the broad applicability of the Cas9 nuclease mediated targeted plant genome editing for development of designer crops. The regulatory uncertainty and social acceptance of plant breeding by Cas9 genome editing have also been described. With this powerful and innovative technique the designer GE non-GM plants could further advance climate resilient and sustainable agriculture in the future and maximizing yield by combating abiotic and biotic stresses.
CRISPR construct design is a key step in the practice of genome editing, which includes identification of appropriate Cas proteins, design and selection of guide RNAs (gRNAs), and selection of ...regulatory elements to express gRNAs and Cas proteins. Here, we review the choices of CRISPR-based genome editors suited for different needs in plant genome editing applications. We consider the technical aspects of gRNA design and the associated computational tools. We also discuss strategies for the design of multiplex CRISPR constructs for high-throughput manipulation of complex biological processes or polygenic traits. We provide recommendations for different elements of CRISPR constructs and discuss the remaining challenges of CRISPR construct optimization in plant genome editing.
Many Cas nucleases (e.g., SpCas9-NRRH, SpG, SpCas9-NG) that can target non-canonical protospacer adjacent motifs (PAMs) have been developed for plant genome editing.Near-PAMless Cas nuclease SpRY has been optimized for plant genome editing to increase the flexibility of gRNA design.A next-generation genome editing technology, prime editing, has been tested in many plants, including Arabidopsis, rice, maize, potato, and tomato.Multiplex clustered regularly interspaced short palindromic repeat (CRISPR) systems based on tRNA/gRNA or Csy4 work better for Cas9 and a hammerhead and hepatitis delta virus (HH-HDV)-based system works better for Cas12a.A multiplex CRISPR system expressing up to 24 gRNAs has been tested in plants.Use of multiple introns in the Cas gene dramatically improves editing efficacy.Improved pegRNA design significantly improves the efficiency of the prime editor.
Targeted DNA insertion in plants Dong, Oliver Xiaoou; Ronald, Pamela C.
Proceedings of the National Academy of Sciences - PNAS,
06/2021, Letnik:
118, Številka:
22
Journal Article
Recenzirano
Odprti dostop
Conventional methods of DNA sequence insertion into plants, using
Agrobacterium
-mediated transformation or microprojectile bombardment, result in the integration of the DNA at random sites in the ...genome. These plants may exhibit altered agronomic traits as a consequence of disruption or silencing of genes that serve a critical function. Also, genes of interest inserted at random sites are often not expressed at the desired level. For these reasons, targeted DNA insertion at suitable genomic sites in plants is a desirable alternative. In this paper we review approaches of targeted DNA insertion in plant genomes, discuss current technical challenges, and describe promising applications of targeted DNA insertion for crop genetic improvement.
CRISPR-Cas systems can be expressed in multiple ways, with different capabilities regarding tissue-specific expression, efficiency, and expression levels. Thus far, three expression strategies have ...been demonstrated in plants: mixed dual promoter systems, dual Pol II promoter systems, and single transcript unit (STU) systems. We explored a fourth strategy to express CRISPR-Cas9 in the model and crop plant, rice, where a bidirectional promoter (BiP) is used to express Cas9 and single guide RNA (sgRNA) in opposite directions. We first tested an engineered BiP system based on double-mini 35S promoter and an
Arabidopsis
enhancer, which resulted in 20.7% and 52.9% genome editing efficiencies at two target sites in T0 stable transgenic rice plants. We further improved the BiP system drastically by using a rice endogenous BiP, OsBiP1. The endogenous BiP expression system had higher expression strength and led to 75.9–93.3% genome editing efficiencies in rice T0 generation, when the sgRNAs were processed by either tRNA or Csy4. We provided a proof-of-concept study of applying BiP systems for expressing two-component CRISPR-Cas9 genome editing reagents in rice. Our work could promote future research and adoption of BiP systems for CRISPR-Cas-based genome engineering in plants.
Plant pathogens pose a major threat to crop productivity. Typically, phytopathogens exploit plants’ susceptibility (S) genes to facilitate their proliferation. Disrupting these S genes may interfere ...with the compatibility between the host and the pathogens and consequently provide broad-spectrum and durable disease resistance. In the past, genetic manipulation of such S genes has been shown to confer disease resistance in various economically important crops. Recent studies have accomplished this task in a transgene-free system using new genome editing tools, including clustered regularly interspaced palindromic repeats (CRISPR). In this Opinion article, we focus on the use of genome editing to target S genes for the development of transgene-free and durable disease-resistant crop varieties.
CRISPR has emerged as a revolutionary tool for plant genome editing. Although developed recently, it has been established in several important plant species, including rice, wheat, and maize, to introduce agronomically important traits such as heat/cold tolerance, disease resistance, herbicide tolerance, and yield improvement.
Transgene-free methods are being introduced in CRISPR-mediated plant genome editing, such as segregating out transgenes, delivering the ribonucleoprotein complex of Cas9 and gRNA through particle bombardment or using a protoplast system, and using viral vectors for editing germline cells.
Targeting susceptibility (S) genes using CRISPR methodologies offers new frontiers to break molecular plant–microbe compatibility and introducing durable pathogen resistance.
Abstract
Genome editing technology is important for plant science and crop breeding. Genome-edited plants prepared using general CRISPR-Cas9 methods usually contain foreign DNA, which is problematic ...for the production of genome-edited transgene-free plants for vegetative propagation or highly heterozygous hybrid cultivars. Here, we describe a method for highly efficient targeted mutagenesis in Nicotiana benthamiana through the expression of Cas9 and single-guide (sg)RNA using a potato virus X (PVX) vector. Following Agrobacterium-mediated introduction of virus vector cDNA, >60% of shoots regenerated without antibiotic selection carried targeted mutations, while ≤18% of shoots contained T-DNA. The PVX vector was also used to express a base editor consisting of modified Cas9 fused with cytidine deaminase to introduce targeted nucleotide substitution in regenerated shoots. We also report exogenous DNA-free genome editing by mechanical inoculation of virions comprising the PVX vector expressing Cas9. This simple and efficient virus vector-mediated delivery of CRISPR-Cas9 could facilitate transgene-free gene editing in plants.
The recent progress in genetic engineering has brought multiple benefits to the food and agricultural industry by enhancing the essential characteristics of agronomic traits. Powerful tools in the ...field of genome editing, such as siRNA-mediated RNA interference for targeted suppression of gene expression and transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs) for DNA repair have been widely used for commercial purposes. However, in the last few years, the discovery of the CRISPR-Cas9 system has revolutionized genome editing and has attracted attention as a powerful tool for several industrial applications. Herein, we review current progresses in the utilization of the CRISPR-Cas9 system in the food and agricultural industry, particularly in the development of resistant crops with improved quality and productivity. We compare the CRISPR system with the TALEN and ZFN nucleases–based methods and highlight potential advantages and shortcomings. In addition, we explore the state of the global market and discuss the safety and ethical concerns associated with the application of this technology in the food and agricultural industry.
•CRISPR faster, easier, more precise and cheaper than traditional genetic methods•CRISPR/Cas9 technology could improve productivity of fermentation processes•No clear mechanism of Cas9 dissociation from designed sgRNA and consequent recycle•Multiple sgRNAs+delivery system open opportunities for gene clusters deletion•Great importance in creating of a global-available database on CRISPR/Cas9 design
Nanomaterial-based delivery systems can deliver functional genes or siRNA into intact plant cells and create transgene-free genetically engineered plants. This system allows highly efficient and ...organelle-specific delivery that can overcome host-range limitations. This approach will have a diverse range of applications in plant biotechnology and plant biology.
Advanced CRISPR-Cas9 based technologies first validated in mammalian cell systems are quickly being adapted for use in plants. These new technologies increase CRISPR-Cas9's utility and effectiveness ...by diversifying cellular capabilities through expression construct system evolution and enzyme orthogonality, as well as enhanced efficiency through delivery and expression mechanisms. Here, we review the current state of advanced CRISPR-Cas9 and Cpf1 capabilities in plants and cover the rapid evolution of these tools from first generation inducers of double strand breaks for basic genetic manipulations to second and third generation multiplexed systems with myriad functionalities, capabilities, and specialized applications. We offer perspective on how to utilize these tools for currently untested research endeavors and analyze strengths and weaknesses of novel CRISPR systems in plants. Advanced CRISPR functionalities and delivery options demonstrated in plants are primarily reviewed but new technologies just coming to the forefront of CRISPR development, or those on the horizon, are briefly discussed. Topics covered are focused on the expansion of expression and delivery capabilities for CRISPR-Cas9 components and broadening targeting range through orthogonal Cas9 and Cpf1 proteins.
Genome editing promises giant leaps forward in advancing biotechnology, agriculture, and basic research. The process relies on the use of sequence specific nucleases (SSNs) to make DNA double ...stranded breaks at user defined genomic loci, which are subsequently repaired by two main DNA repair pathways: non-homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ can result in frameshift mutations that often create genetic knockouts. These knockout lines are useful for functional and reverse genetic studies but also have applications in agriculture. HDR has a variety of applications as it can be used for gene replacement, gene stacking, and for creating various fusion proteins. In recent years, transcription activator-like effector nucleases and clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR associated protein 9 or CRISPR from Prevotella and Francisella 1 have emerged as the preferred SSNs for research purposes. Here, we review their applications in plant research, discuss current limitations, and predict future research directions in plant genome editing.
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