Rapid developments in the field of plant genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems necessitate more detailed consideration of the delivery of the CRISPR system into plants. Successful and safe editing of plant genomes is partly based on efficient delivery of the CRISPR system. Along with the use of plasmids and viral vectors as cargo material for genome editing, non-viral vectors have also been considered for delivery purposes. These non-viral vectors can be made of a variety of materials, including inorganic nanoparticles, carbon nanotubes, liposomes, and protein- and peptide-based nanoparticles, as well as nanoscale polymeric materials. They have a decreased immune response, an advantage over viral vectors, and offer additional flexibility in their design, allowing them to be functionalized and targeted to specific sites in a biological system with low cytotoxicity. This review is dedicated to describing the delivery methods of CRISPR system into plants with emphasis on the use of non-viral vectors.
The revolutionary technology of CRISPR/Cas systems and their extraordinary potential to address fundamental questions in every field of biological sciences has led to their developers being awarded the 2020 Nobel Prize for Chemistry. In agriculture, CRISPR/Cas systems have accelerated the development of new crop varieties with improved traits—without the need for transgenes. However, the future of this technology depends on a clear and truly global regulatory framework being developed for these crops. Some CRISPR-edited crops are already on the market, and yet countries and regions are still divided over their legal status. CRISPR editing does not require transgenes, making CRISPR crops more socially acceptable than genetically modified crops, but there is vigorous debate over how to regulate these crops and what precautionary measures are required before they appear on the market. This article reviews intended outcomes and risks arising from the site-directed nuclease CRISPR systems used to improve agricultural crop plant genomes. It examines how various CRISPR system components, and potential concerns associated with CRISPR/Cas, may trigger regulatory oversight of CRISPR-edited crops. The article highlights differences and similarities between GMOs and CRISPR-edited crops, and discusses social and ethical concerns. It outlines the regulatory framework for GMO crops, which many countries also apply to CRISPR-edited crops, and the global regulatory landscape for CRISPR-edited crops. The article concludes with future prospects for CRISPR-edited crops and their products.
Fish is a fundamentally healthy food, loaded with essential nutrients, high protein content, vitamin D, and omega-three fatty acid. Mislabeling is a common problem in the fish industry that causes an imbalance in prices and fluctuation in the market. DNA barcoding is a potential technique for authentication of mislabeled and misidentified fish species. In this study, 11 freshwater and 6 marine fish species were used for DNA barcoding and further authentication using the mitochondrial cytochrome b (Cyt b) gene. Cyt b was amplified using PCR, producing an average read length of 1,141 bp. The obtained sequences were compared to the National Center for Biotechnology Information database (NCBI) using the Basic Local Alignment Search Tool (BLAST). The average AT content (55.20%) was higher than the average GC content (44.78%) in marine and freshwater fish species. The mean genetic Kimura 2-parameter distances for species, genus, families, and orders were 0.311, 0.308, 0.023, and 0.337, respectively. Phylogenetic tree analysis revealed that most of the freshwater fish species clustered together due to the fact that they were in the same order or family, while the marine fish species clustered distantly. Single nucleotide polymorphism (SNP) analysis of all species in the study revealed distinct features regarding unique sites. All fish species could be identified based on their unique SNP profiles. Based on SNP data, DNA sequence based QR codes were developed for accurate identification of fish species. This is the first study to develop DNA-based QR barcodes for proper authentication of species during the chain of custody using simple technology.
Engineered nucleases have emerged as a powerful tool for site specific gene manipulation in plants. Based on Clustered Regularly Interspersed Short Palindromic Repeats/CRISPR associated (CRISPR/Cas) system, engineered Cas9:gRNA complex can be used to cleave specific DNA sequences in the genome. In the present study, Nicotiana benthamiana Phytoene Desaturase (NbPDS) gene was targeted by CRISPR/Cas9 system. The plant codon optimized (pcoCas9) along with guided RNA (gRNA) was cloned in plant expression vector pGreen0029, to target NbPDS gene. The NbPDS gene was disrupted transiently by agroinfiltration of pcoCas9-gRNA complex. Visible albino spots were observed on agro-infiltrated leaves of N. benthamiana plants after 7 days of infiltration. The observed albino spots were analyzed through PCR amplification of gRNAtarget, fluorescent microscopy and chlorophyll contents measurements. Our results support the notion that CRISPR/Cas9 system is a swift, robust and useful tool for targeted gene disruption, deletion and editing.
Conventional tools induce mutations randomly throughout the cotton genome-making breeding difficult and challenging. During the last decade, progress has been made to edit the gene of interest in a very precise manner. Targeted genome engineering with engineered nucleases (ENs) specifically zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases (e.g., Cas9) has been described as a "game-changing technology" for diverse fields as human genetics and plant biotechnology. In eukaryotic systems, ENs create double-strand breaks (DSBs) at the targeted DNA sequence which are repaired by nonhomologous end joining (NHEJ) or homologydirected recombination (HDR) mechanisms. ENs have been used successfully for targeted mutagenesis, gene knockout, and multisite genome editing (GenEd) in model plants and crop plants such as cotton, rice, and wheat. Recently, cotton genome has also been edited for targeted mutagenesis through CRISPR/Cas for improved lateral root formation. In addition, an efficient and fast method has been developed to evaluate guide RNAs transiently in cotton. The targeted disruption of undesirable genes or metabolic pathway can be achieved to increase quality of cotton. Undesirable metabolites like gossypol in cottonseed can be targeted efficiently using ENs for seed-specific low-gossypol cotton. Moreover, ENs are also helpful in gene stacking for herbicide resistance, insect resistance, and abiotic stress tolerance.
Cotton is a major cash crop and backbone of the textile industry in Pakistan which is badly affected by sucking insects. Drought is an important abiotic factor in trichome development. The objective of the study was to determine the effects of drought on trichome density and length. Trichome density was measured in two ways, one through the scaling method and the other through counting the trichome density manually. The scaling method is qualitative grading while quantitative grading includes trichomecount in a card of optimized length. Three scales were finalized to classify leaves on the basis of trichomes which were counted in a specific area (0.25cm2) on abaxial side of the leaf. In drought stress, trichomes density and length were measured and compared to that in normal conditions. Trichome density varies from 12 to 56 in 0.25cm2 under drought stress. On the basis of correlation of trichome density with stomatal conductance, photosynthetic rate, PAR and transpiration ratio under drought and normal conditions, it was concluded that trichome density increased as a result of drought stress.
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