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Unlocking the potential of CRISPR technology for improving livelihoods in Africa.
Africa is burdened with food shortages and plant, animal and human diseases. Some of these can be ameliorated by adopting genome editing technologies such as CRISPR. This technology is considered better than its predecessors, Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), because it is cheaper, easy to use, has high gene modification efficiency and is less time consuming. CRISPR technology has wide applications in the African context ranging from crop and animal improvement to disease diagnosis and treatment as well as improving food shelf life, organoleptic properties and food safety. It has the potential to bring back species of organisms that are extinct. However, some African countries have not taken advantage of the potential of CRISPR to solve many of their problems. This paper explores possible applications of CRISPR towards improvement of African livelihoods.
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Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair.
Non-homologous end-joining (NHEJ) plays an important role in double-strand break (DSB) repair of DNA. Recent studies have shown that the error patterns of NHEJ are strongly biased by sequence context, but these studies were based on relatively few templates. To investigate this more thoroughly, we systematically profiled ∼1.16 million independent mutational events resulting from CRISPR/Cas9-mediated cleavage and NHEJ-mediated DSB repair of 6872 synthetic target sequences, introduced into a human cell line via lentiviral infection. We find that: (i) insertions are dominated by 1 bp events templated by sequence immediately upstream of the cleavage site, (ii) deletions are predominantly associated with microhomology and (iii) targets exhibit variable but reproducible diversity with respect to the number and relative frequency of the mutational outcomes to which they give rise. From these data, we trained a model that uses local sequence context to predict the distribution of mutational outcomes. Exploiting the bias of NHEJ outcomes towards microhomology mediated events, we demonstrate the programming of deletion patterns by introducing microhomology to specific locations in the vicinity of the DSB site. We anticipate that our results will inform investigations of DSB repair mechanisms as well as the design of CRISPR/Cas9 experiments for diverse applications including genome-wide screens, gene therapy, lineage tracing and molecular recording.
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CRISPR/Cas9 for Mutagenesis in Rice.
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) provides a workhorse for genome editing biotechnology. CRISPR/Cas9 tailored for enabling genome editing has been extensively interrogated and widely utilized for precise genomic alterations in eukaryotic organisms including in plant species. The technology holds the great promise to better understand gene functions, elucidate networks, and improve the performance of crop plants such as increasing grain yields, improving nutritional content, and better combating the biotic and abiotic stresses. Various methods or protocols specific for different plant species have been established. Here, we present a CRISPR/Cas9-mediated genome editing protocol in rice, including detailed information about single-guide RNA design, vector construction, plant transformation, and mutant screening processes.
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CRISPR/Cas9 Technology and Its Utility for Crop Improvement.
The rapid growth of the global population has resulted in a considerable increase in the demand for food crops. However, traditional crop breeding methods will not be able to satisfy the worldwide demand for food in the future. New gene-editing technologies, the most widely used of which is CRISPR/Cas9, may enable the rapid improvement of crop traits. Specifically, CRISPR/Cas9 genome-editing technology involves the use of a guide RNA and a Cas9 protein that can cleave the genome at specific loci. Due to its simplicity and efficiency, the CRISPR/Cas9 system has rapidly become the most widely used tool for editing animal and plant genomes. It is ideal for modifying the traits of many plants, including food crops, and for creating new germplasm materials. In this review, the development of the CRISPR/Cas9 system, the underlying mechanism, and examples of its use for editing genes in important crops are discussed. Furthermore, certain limitations of the CRISPR/Cas9 system and potential solutions are described. This article will provide researchers with important information regarding the use of CRISPR/Cas9 gene-editing technology for crop improvement, plant breeding, and gene functional analyses.
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CRISPR/Cas9-assisted gene editing reveals that EgPKS, a polyketide synthase, is required for the biosynthesis of preussomerins in Edenia gomezpompae SV2.
Edenia gomezpompae, an endophytic fungus derived from plants, produced a diverse array of preussomerins, a type of spirobisnaphthalenes featuring two spiroketal groups, which exhibited significant antibacterial, antifungal, and cytotoxic activities. Structurally, the biosynthesis of preussomerins might be related to the biosynthesis of 1,8-dihydroxynaphthalene (DHN), a precursor of DHN-melanin. However, the absence of efficient gene-editing tools for E. gomezpompae has hindered the biosynthetic study of preussomerins. In this study, we developed a CRISPR/Cas9-based gene editing system for E. gomezpompae SV2 that was isolated from the stem of Setaria viridis, by utilizing the endogenous U6 snRNA promoter to drive sgRNA expression. Using this system, we successfully disrupted the polyketide synthase (PKS)-encoding gene, Egpks, a putative 1,3,6,8-tetrahydroxynaphthalene synthase gene involved in the biosynthesis of DHN-melanin, with an editing efficiency up to 92% and a knockout efficiency of 71% when employing the U6 snRNA-3 promoter. Furthermore, the disrupted mutant (∆Egpks) displayed white hyphae and lost the ability to produce preussomerins. These results provided a foundational tool for genetic manipulation in E. gomezpompae and revealed the role of EgPKS in the biosynthesis of preussomerin-type spirobisnaphthalenes.
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Lipopolymeric Nanocarrier Enables Effective Delivery of CRISPR/Cas9 Expressing Plasmid.
CRISPR/Cas9 has proven its accuracy and precision for gene editing by making a double-strand break at the predetermined site. Despite being a mainstream gene editing tool, CRISPR/Cas9 has limitations for its in vivo delivery due to the physico-chemical properties such as high molecular weight, supranegative charge, degradation in the presence of nucleases, etc. Hereby, a cationic lipopolymer is explored for its efficiency in delivering CRISPR/Cas9 plasmid (pCas9) in vitro and in vivo. The lipopolymer is utilized to form blank cationic nanoplexes having a zeta potential of +15.8 ± 0.7 mV. Being cationic, the blank nanoplexes are able to condense the pCas9 plasmid at a ratio of 1:20 with a complexation efficiency of ≈98% and show a size and zeta potential of ≈141 ± 16 nm and 4.2 mV ± 0.7, respectively. The pCas9-loaded nanoplexes show a transfection efficiency of ≈69% in ARPE-19 cells and show ≈22% of indel frequency, indicating the successful translation of Cas9 protein and guide RNA in the cytosol. Further, they are found to be stable under in vivo environment when given intravenously in Swiss albino mice. These lipopolymeric nanoplexes can be a potential carrier for CRISPR plasmids for genome editing applications.
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Harnessing the potential of gene editing technology using CRISPR in inflammatory bowel disease.
The molecular scalpel of clustered regularly interspersed short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) technology may be sharp enough to begin cutting the genes implicated in inflammatory bowel disease (IBD) and consequently decrease the 6.3 billion dollar annual financial healthcare burden in the treatment of IBD. For the past few years CRISPR technology has drastically revolutionized DNA engineering and biomedical research field. We are beginning to see its application in gene manipulation of sickle cell disease, human immunodeficiency virus resistant embryologic twin gene modification and IBD genes such as Gatm (Glycine amidinotransferase, mitochondrial), nucleotide-binding oligomerization domain-containing protein 2, KRT12 and other genes implicated in adaptive immune convergence pathways have been subjected to gene editing, however there are very few publications. Furthermore, since Crohn's disease and ulcerative colitis have shared disease susceptibility and share genetic gene profile, it is paramount and is more advantageous to use CRISPR technology to maximize impact. Although, currently CRISPR does have its limitations due to limited number of specific Cas enzymes, off-target activity, protospacer adjacent motifs and crossfire between different target sites. However, these limitations have given researchers further insight on how to augment and manipulate enzymes to enable precise gene excision and limit crossfire between target sites.
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Nicotine-free, nontransgenic tobacco (Nicotiana tabacum l.) edited by CRISPR-Cas9.
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CRISPR Technology for Livestock Improvement: Advances and Future Directions.
CRISPR-Cas9 technology is revolutionizing genetic engineering by enabling precise genome modifications across a wide range of organisms, particularly in livestock. This review focuses on livestock improvement and the most recent transformative developments in the application of CRISPR-based genome editing techniques. We analyzed productivity improvements achieved by editing growth factor genes, immunogenic resistance enhancement through the editing of immune loci, and ecological footprint reduction for sustainability. This review also addresses the intricate ethical and regulatory issues posed by the application of CRISPR technology in animal breeding, including concerns about animal welfare violations, unintended off-target effects, and impacts on public perception. Furthermore, we discuss key untapped CRISPR targets in livestock genetics enabled by precision gene editing, the emerging integration of CRISPR with artificial intelligence, and the need for collaboration to address complex ethical and regulatory challenges related to applying CRISPR in animal breeding.
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Emerging CRISPR Therapies for Precision Gene Editing and Modulation in the Cardiovascular Clinic.
PURPOSE OF REVIEW: Outline the growing suite of novel genome editing tools powered by CRISPR-Cas9 technology that are rapidly advancing towards the clinic for the treatment of cardiovascular disorders. RECENT FINDINGS: A diversity of new genome editors and modulators are being developed for therapies across myriad human diseases. Recent breakthroughs have improved the efficacy, safety, specificity, and delivery of CRISPR-mediated therapies that could impact heart disease in the next decade, though several challenges remain. Many iterations of the original CRISPR system have been developed seeking to leverage its vast therapeutic potential. As examples, nuclease-free editing, precision single-nucleotide editing, gene expression regulation, and epigenomic modifications are now feasible with the current CRISPR-mediated suite of enzymes. These emerging tools will be indispensable for the development of novel cardiovascular therapeutics as demonstrated by recent successes in both basic research laboratories and pre-clinical models. Here, we provide an overview of current and emerging CRISPR-mediated technologies as they pertain to the cardiovascular system, highlighting successful implementations and future challenges.
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