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Plant Tissue Culture: A Battle Horse in the Genome Editing Using CRISPR/Cas9.
Plant tissue culture (PTC) is a set of techniques for culturing cells, tissues, or organs in an aseptic medium with a defined chemical composition, in a controlled environment. Tissue culture, when combined with molecular biology techniques, becomes a powerful tool for the study of metabolic pathways, elucidation of cellular processes, genetic improvement and, through genetic engineering, the generation of cell lines resistant to biotic and abiotic stress, obtaining improved plants of agronomic interest, or studying the complex cellular genome. In this chapter, we analyze in general the use of plant tissue culture, in particular protoplasts and calli, in the implementation of CRISPR/Cas9 technology.
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Synergizing CRISPR-Cas9 with Advanced Artificial Intelligence and Machine Learning for Precision Drug Delivery: Technological Nexus and Regulatory Insights.
The evolution of genetic exploration tools, from laborious methods like radiationinduced mutations to the transformative CRISPR-Cas9 system, has fundamentally reshaped genetic research and gene editing capabilities. This journey, initiated by foundational techniques such as ZFNs and TALENs and culminating in the groundbreaking work of Doudna and Charpentier in 2012, has ushered in an era of precise DNA alteration and profound insights into gene functions. The CRISPR/Cas9 system uses the Cas9 enzyme and guides RNA (gRNA) to precisely target and cleave DNA, with subsequent repair via error-prone NHEJ or precise HDR, enabling versatile gene editing. Complementary computational tools like E-CRISP and Azimuth 2.0, alongside advanced deep learning models like DeepCRISPR, have significantly contributed to refining CRISPR experiments, optimizing gRNA efficiency, and predicting outcomes with greater precision. In clinical applications, CRISPR-Cas9 shows great promise for treating complex genetic disorders like sickle cell disease and β-thalassemia, but faces challenges such as off-target effects, immune responses to viral vectors, and ethical issues in germline editing. Overcoming these challenges requires meticulous experimentation and robust regulatory frameworks to ensure responsible and beneficial utilization of the CRISPR-Cas9 technology across diverse fields, including cancer treatment, genetic disease therapies, agriculture, and synthetic biology, while continually addressing ethical, safety, and legal considerations for its advancement and widespread adoption.
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CRISPR/Cas9 in soybean research: a measured leap toward genetic refinement.
Soybean [Glycine max (Linn.) Merr] is a globally important source of plant-based protein and edible oil, widely cultivated in India as a major oilseed crop. However, conventional breeding approaches for soybean are laborious, constrained by the genomic complexity of soybean, and face persistent challenges in genetic transformation. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-based genome editing has emerged as a precise and efficient tool for rapid trait improvement in soybean. While many existing reviews of soybean have been published, this literature review addresses some fundamental challenges with respect to genome editing. This study summarizes recent advancements in soybean genome editing, offering insights into the practical informatics of soybean-specific genomic databases that are required for successful construct design. It also discusses emerging technologies such as base editing, prime editing, and the increasingly important role of artificial intelligence in accelerating target identification and construct design. It highlights applications in nutritional enhancement, abiotic and biotic stress tolerance, and improvement of key agronomic traits. Beyond the conventional topics, it addresses the current regulatory landscape that researchers must navigate for releasing genome-edited varieties and provides a comprehensive perspective on practical solutions to overcome major constraints in soybean genetic engineering.
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Advances in CRISPR/Cas9.
CRISPR/Cas9 technology has become the most examined gene editing technology in recent years due to its simple design, yet low cost, high efficiency, and simple operation, which can also achieve simultaneous editing of multiple loci. It can also be carried out without using plasmids, saving lots of troubles caused by plasmids. CRISPR/Cas9 has shown great potential in the study of genes or genomic functions in microorganisms, plants, animals, and human beings. In this review, we will examine the history, structure, and basic mechanisms of the CRISPR/Cas9 system, describe its great value in precision medicine and sgRNA library screening, and dig its great potential in a new field: DNA information storage.
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Generation of albino C57BL/6J mice by CRISPR embryo editing of the mouse tyrosinase locus.
After the arrival of the CRISPR/Cas9 genome editing technology, genetic engineering of model organisms has become much faster and more efficient. The development of genetically modified mouse models is also facilitated by the application of various CRISPR methodologies. Although the very first studies utilized pronuclear injection (PNI) of Cas9 mRNA and sgRNAs into the zygote stage embryos to create knockout and knockin mutations, the repertoire of techniques and collection of reagents for CRISPR editing has rapidly expanded. This presents researchers in the field with a versatility of choices for genetic engineering. However, there are not many comparative studies that analysed the efficacy of gene editing when Cas9 and sgRNA/ssDNA oligos were transferred to the embryos by different methodologies. Here, we aimed to compare two different methods, electroporation and PNI. One of the recent developments gaining wide use in mouse model research is the application of electroporation for the introduction of Cas9/sgRNA ribonucleoprotein complexes into zygote stage embryos. Here, we have used this technique to generate albino coat-coloured C57BL/6J mice by targeted inactivation of the mouse tyrosinase gene through indel or knockin mutations. We have also applied the PNI protocol with the same set of reagents, to compare the efficiency of the two techniques in generation of indel and knockin mutations. Although PNI results in signifi- cantly higher efficiency for knockin mutations, it requires specialized equipment setup and advanced training in embryo micromanipulation and microinjection. Therefore, for the generation of simple gene knockouts by indel mutations, electroporation can be used.
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The use of plant-derived exosome-like nanoparticles as a delivery system of CRISPR/Cas9-based therapeutics for editing long non-coding RNAs in cancer colon cells.
Colon cancer is one of the leading causes of cancer in the United States. Colon cancer develops from the many gene mutations found in the genomes of colon cancer cells. Long non-coding RNAs (lncRNAs) can cause the development and progression of many cancers, including colon cancer. LncRNAs have been and could be corrected through the gene-editing technology of the clustered repeats of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (CRISPR/Cas9) system to reduce the proliferation of cancer cells in the colon. However, many current delivery systems for transporting CRISPR/Cas9-based therapeutics in vivo need more safety and efficiency. CRISPR/Cas9-based therapeutics require a safe and effective delivery system to more directly and specifically target cancer cells present in the colon. This review will present pertinent evidence for the increased efficiency and safety of using plant-derived exosome-like nanoparticles as nanocarriers for delivering CRISPR/Cas9-based therapeutics to target colon cancer cells directly.
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Gene editing technology: Towards precision medicine in inherited retinal diseases.
Purpose: To review preclinical and clinical advances in gene therapy, with a focus on gene editing technologies, and application to inherited retinal disease.Methods: A narrative overview of the literature, summarizing the state-of-the-art in clinical gene therapy for inherited retinal disease, as well as the science and application of new gene editing technology.Results: The last three years has seen the first FDA approval of an in vivo gene replacement therapy for a hereditary blinding eye disease and, recently, the first clinical application of an in vivo gene editing technique. Limitations and challenges in this evolving field are highlighted, as well as new technologies developed to address the multitude of molecular mechanisms of disease.Conclusion: Genetic therapy for the treatment of inherited retinal disease is a rapidly expanding area of ophthalmology. New technologies have revolutionized the field of genome engineering and rekindled an interest in precision medicines for these conditions.
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China's approval of gene-edited crops energizes researchers.
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Hydroxysteroid sulfotransferase 2B1 affects gastric epithelial function and carcinogenesis induced by a carcinogenic agent.
BACKGROUND: A healthy gastric mucosal epithelium exhibits tumor-suppressive properties. Gastric epithelial cell dysfunction contributes to gastric cancer development. Oxysterols provided from food or cholesterol oxidation in the gastric epithelium may be further sulfated by hydroxysteroid sulfotransferase 2B1 (SULT2B1), which is highly abundant in the gastric epithelium. However, the effects of SULT2B1 on gastric epithelial function and gastric carcinogenesis are unclear. METHODS: A mouse gastric tumor model was established using carcinogenic agent 3-methylcholanthrene (3-MCA). A SULT2B1 deletion (SULT2B1-/-) human gastric epithelial line GES-1 was constructed by CRISPR/CAS9 genome editing system. RESULTS: The gastric tumor incidence was higher in the SULT2B1-/- mice than in the wild-type (WT) mice. In gastric epithelial cells, adenovirus-mediated SULT2B1b overexpression reduced the levels of oxysterols, such as 24(R/S),25-epoxycholesterol (24(R/S),25-EC) and 27-hydroxycholesterol (27HC). This condition also increased PI3K/AKT signaling to promote gastric epithelial cell proliferation, epithelization, and epithelial development. However, SULT2B1 deletion or SULT2B1 knockdown suppressed PI3K/AKT signaling, epithelial cell epithelization, and wound healing and induced gastric epithelial cell malignant transition upon 3-MCA induction. CONCLUSIONS: The abundant SULT2B1 expression in normal gastric epithelium might maintain epithelial function via the PI3K/AKT signaling pathway and suppress gastric carcinogenesis induced by a carcinogenic agent.
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Mosaicism in CRISPR/Cas9-mediated genome editing.
The CRISPR/Cas9 system is a rapid, simple, and often extremely efficient gene editing method. This method has been used in a variety of organisms and cell types over the past several years. However, using this technology for generating gene-edited animals involves a number of obstacles. One such obstacle is mosaicism, which is common in founder animals. This is especially the case when the CRISPR/Cas9 system is used in embryos. Here we review the pros and cons of mosaic mutations of gene-edited animals caused by using the CRISPR/Cas9 system in embryos. Furthermore, we will discuss the mechanisms underlying mosaic mutations resulting from the CRISPR/Cas9 system, as well as the possible strategies for reducing mosaicism. By developing ways to overcome mosaic mutations when using CRISPR/Cas9, genotyping for germline gene disruptions should become more reliable. This achievement will pave the way for using the CRISPR technology in the research and clinical applications where mosaicism is an issue.
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