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Roles of TP53 gene in the development of resistance to PI3K inhibitor resistances in CRISPR-Cas9-edited lung adenocarcinoma cells.
The mutation rates of tumor suppressor protein p53 gene (TP53) are high in lung adenocarcinoma and promote the development of acquired drug resistance. The present study evaluated the p53-dependent role in lung cancer cell sensitivity to PI3K-specific inhibitors, PI3K-associated inhibitors, PI3K-non-related inhibitors, and protein-based stimuli using designed p53 mutation. We found that the deletion of p53 key regions from amino acid 96 to 393 with the CRISPR-Cas9 altered multi-dimensional structure and sequencing of p53, probably leading the secondary changes in chemical structures and properties of PI3K subunit proteins or in interactions between p53 and PI3K isoform genes. The p53-dependent cell sensitivity varied among target specificities, drug chemical properties, mechanism-specific signal pathways, and drug efficacies, independently upon the size of molecules. The effects of the designed p53 mutation highly depend upon p53-involved molecular mechanisms in the cell. Our results indicate that lung cancer cell resistance to drug can develop with dynamic formations of p53 mutations changing the cell sensitivity. This may explain the real-time occurrence of cancer cell resistance to drug treatment, during which drugs may induce the new mutations of p53. Thus, it is important to dynamically monitor the formation of new mutations during the therapy and discover new drug resistance-specific targets.
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Use of designer nucleases for targeted gene and genome editing in plants.
The ability to efficiently inactivate or replace genes in model organisms allowed a rapid expansion of our understanding of many of the genetic, biochemical, molecular and cellular mechanisms that support life. With the advent of new techniques for manipulating genes and genomes that are applicable not only to single-celled organisms, but also to more complex organisms such as animals and plants, the speed with which scientists and biotechnologists can expand fundamental knowledge and apply that knowledge to improvements in medicine, industry and agriculture is set to expand in an exponential fashion. At the heart of these advancements will be the use of gene editing tools such as zinc finger nucleases, modified meganucleases, hybrid DNA/RNA oligonucleotides, TAL effector nucleases and modified CRISPR/Cas9. Each of these tools has the ability to precisely target one specific DNA sequence within a genome and (except for DNA/RNA oligonucleotides) to create a double-stranded DNA break. DNA repair to such breaks sometimes leads to gene knockouts or gene replacement by homologous recombination if exogenously supplied homologous DNA fragments are made available. Genome rearrangements are also possible to engineer. Creation and use of such genome rearrangements, gene knockouts and gene replacements by the plant science community is gaining significant momentum. To document some of this progress and to explore the technology's longer term potential, this review highlights present and future uses of designer nucleases to greatly expedite research with model plant systems and to engineer genes and genomes in major and minor crop species for enhanced food production.
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Efficient gene editing of BMP15, GDF9, and MSTN-but not the imprinted CLPG gene-in goat embryos via electrotransfection and handmade cloning.
CRISPR/Cas9 technology represents a powerful tool for advancing livestock breeding by enabling precise, on-target gene edits without the genomic mixing associated with traditional introgression methods. In this study, we employed a dual gRNA-based CRISPR/Cas9 strategy to induce targeted deletions and indel mutations in both reproductive and growth-related genes. These included the metacentric genes bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), which are associated with increased ovulation rate and litter size, as well as the telomeric genes myostatin (MSTN) and callipyge (CLPG), which are linked to muscle development and enhanced meat production. We employed an optimized electrotransfection protocol consisting of 10-20 µg of each plasmid DNA, 250 µL OptiMEM-GlutaMAX, and one million goat fibroblast cells. The electroporation was performed using a Bio-Rad system in a 4-mm cuvette, with two 10-millisecond pulses at 270 volts, separated by a 10-second interval. This protocol enabled efficient genome editing of goat embryonic fibroblast cells, which were subsequently used to generate cloned embryos via handmade somatic cell nuclear transfer (SCNT), involving manual enucleation and cell-oocyte fusion steps. Sequencing revealed high mutation rates (78-97%) and a predominance of biallelic edits in BMP15, GDF9, and MSTN. Notably, MSTN gRNAs with a 7-bp overlapping sequence at their 3' ends showed a high editing efficiency. In contrast, the imprinted CLPG gene exhibited a significantly lower mutation rate (~ 30%), likely due to epigenetic constraints. While overall mutation rates did not differ significantly between metacentric and telomeric genes, on-target deletions were more frequent in metacentric genes (43%) than in telomeric ones (20%). Embryo development rates from gene-edited cells were comparable to those from non-edited controls. These findings underscore the utility of combining electrotransfection with SCNT for efficient editing of non-imprinted genes and highlight the need for improved strategies to overcome barriers in editing imprinted loci.
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An efficient genetic transformation and gene editing system mediated by Agrobacterium rhizogenes for Liriodendron hybrid and its application.
An efficient genetic transformation system is imperative for advancing gene functional studies and molecular breeding in horticultural tree species. Traditional Agrobacterium tumefaciens-mediated methods have encountered significant technical challenges when applied to tree species, particularly Liriodendron hybrids. The high costs, and extended transformation cycles associated with this method have substantially limited its utility in gene functional analysis and breeding applications. In response to these challenges, this study presents the development of a streamlined, rapid, and efficient Agrobacterium rhizogenes-mediated transformation system tailored specifically for Liriodendron hybrids, an important ornamental and timber tree species. Among the three Agrobacterium rhizogenes strains (K599, MSU440, and C58C1) evaluated in this study, K599 demonstrated the highest transformation efficiency, reaching 46.09 % for inducing hairy roots from apical bud incisions. Further optimization of the system revealed that 2 days and two-month-old seedlings were the most suitable co-culture duration and explants, yielding a peak transformation efficiency of 60.38 %, 53.12 %, respectively. The applicability of this transformation system was validated across various Liriodendron hybrids genotypes and Liriodendron tulipifera, with transformation efficiencies ranging from 15.47 % to 60.63 %. Additionally, the system was effectively employed for subcellular localization analysis, which confirmed that the aquaporin (AQP) protein. LhAQP1 is localized in the plasma membrane and exhibits enrichment in the vascular tissues of the hairy roots. Notably, this study marks the first application of the Agrobacterium rhizogenes-mediated system for CRISPR/Cas9-mediated gene editing in Liriodendron hybrids, successfully achieving targeted mutagenesis of the LhAQP1 gene and establishing the feasibility of gene editing within this species. In addition, the hairy root-based transformation system was employed to investigate the functional role of LhAQP1 in the improved Liriodendron variety 'Nanlin-Jinsen E1' by comparing wild-type, LhAQP1-overexpressing and gene-edited lines generated via the CRISPR/Cas9. LhAQP1-overexpression significantly promoted plant growth and drought tolerance, whereas pYLCRISPR-LhAQP1 increases dehydration sensitivity, underscoring the essential role of LhAQP1 in water stress adaptation. Optimized transformation platform represents a crucial advancement for functional genomics and molecular breeding efforts in woody plants.
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Gene Editing of Primary Rhesus Macaque B Cells.
B cells and their progeny are the sources of highly expressed antibodies. Their high protein expression capabilities together with their abundance, easy accessibility via peripheral blood, and amenability to simple adoptive transfers have made them an attractive target for gene editing approaches to express recombinant antibodies or other therapeutic proteins. The gene editing of mouse and human primary B cells is efficient, and mouse models for in vivo studies have shown promise, but feasibility and scalability for larger animal models have so far not been demonstrated. We, therefore, developed a protocol to edit rhesus macaque primary B cells in vitro to enable such studies. We report conditions for in vitro culture and gene-editing of primary rhesus macaque B cells from peripheral blood mononuclear cells or splenocytes using CRISPR/Cas9. To achieve the targeted integration of large (<4.5 kb) cassettes, a fast and efficient protocol was included for preparing recombinant adeno-associated virus serotype 6 as a homology-directed repair template using a tetracycline-enabled self-silencing adenoviral helper vector. These protocols enable the study of prospective B cell therapeutics in rhesus macaques.
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Cell Synchronization Enhances Nuclear Transformation and Genome Editing via Cas9 Enabling Homologous Recombination in Chlamydomonas reinhardtii.
In Chlamydomonas reinhardtii, the model organism for eukaryotic green algae and plants, the processes of nuclear transformation and genome editing in particular are still marked by a low level of efficiency, and so intensive work is required in order to create and identify mutants for the investigation of basic physiological processes, as well as the implementation of biotechnological applications. In this work, we show that cell synchronization during the stages of the cell cycle, obtained from long-term cultivation under specific growth conditions, greatly enhances the efficiency of transformation and allows the identification of DNA repair mechanisms that occur preferentially at different stages of the cell cycle. We demonstrate that the transformation of synchronized cells at different times was differentially associated with nonhomologous end joining (NHEJ) and/or homologous recombination (HR), and makes it possible to knock-in specific foreign DNA at the genomic nuclear location desired by exploiting HR. This optimization greatly reduces the overall complexity of the genome editing procedure and creates new opportunities for altering genes and their products.
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Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system.
Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6 % for the wild-type strain and 94.1 % for the ku70/ku80 double-deficient strain, within 4 days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3 %, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica.
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Engineering and comparison of cas12a-based genome editing systems in plants.
While Cas9 and Cas12a are both RNA-guided endonucleases used for genome editing, only Cas12a is able to process pre-crRNA via its additional ribonuclease activity. This feature reduces the complexity of Cas12a versus Cas9-based genome editing systems thus providing an attractive alternative for generating site-specific mutations in plants. Here we aimed to improve the efficiency of the cas12a-based generation of two double-strand breaks flanking the open reading frame of a target gene, leading to its full deletion. To this end, we compared the relative impact of different components on cas12a-based gene deletion efficiency in three different eudicotyledons, Arabidopsis thaliana, Lotus japonicus, and Nicotiana benthamiana. We detected the highest cas12a-based editing efficiency with a combination of suitable promoters for crRNA and cas12a expression, a tandem terminator to control cas12a expression, a re-coded cas12a, adapted to the codon usage of Arabidopsis and engineered to carry introns, and encoding a Cas12a flanked by a nuclear localization signal at both ends. Our work revealed the high potential for improving cas12a-based genome editing systems for plant genetic research.
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CRISPR/Cas9 Gene Editing of RYR2 in Human iPSC-Derived Cardiomyocytes to Probe Ca2+ Signaling Aberrancies of CPVT Arrhythmogenesis.
Human-induced pluripotent stem cells (hiPSCs) provide a powerful platform to study biophysical and molecular mechanisms underlying the pathophysiology of genetic mutations associated with cardiac arrhythmia. Human iPSCs can be generated by reprograming of dermal fibroblasts of normal or diseased individuals and be differentiated into cardiac myocytes. Obtaining biopsies from patients afflicted with point mutations causing arrhythmia is often a cumbersome process even when patients are available. Recent development of CRISPR/Cas9 gene editing system makes it, however, possible to introduce arrhythmia-associated point mutations at the desired loci of the wild-type hiPSCs in relatively short times. This platform was used by us to compare the Ca2+ signaling phenotypes of cardiomyocytes harboring point mutations in cardiac Ca2+ release channel, type-2 ryanodine receptor (RyR2), since over 200 missense mutations in RYR2 gene appear to be associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1). We have created cardiac myocytes harboring mutations in different domains of RyR2, to study not only their Ca2+ signaling consequences but also their drug and domain specificity as related to CPVT1 pathology. In this chapter, we describe our procedures to establish CRISPR/Cas9 gene-edited hiPSC-derived cardiomyocytes.
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Enhancing CRISPR/Cas9-mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52.
Precise genome editing with desired point mutations can be generated by CRISPR/Cas9-mediated homology-directed repair (HDR) and is of great significance for gene function study, gene therapy and animal breeding. However, HDR efficiency is inherently low and improvements are necessitated. Herein, we determined that the HDR efficiency could be enhanced by expressing Rad52, a gene that is involved in the homologous recombination process. Both the Rad52 co-expression and Rad52-Cas9 fusion strategies yielded approximately 3-fold increase in HDR during the surrogate reporter assays in human HEK293T cells, as well as in the genome editing assays. Moreover, the enhancement effects of the Rad52-Cas9 fusion on HDR mediated by different (plasmid, PCR and ssDNA) donor templates were confirmed. We found that the HDR efficiency could be significantly improved to about 40% by the combined usage of Rad52 and Scr7. In addition, we also applied the fusion strategy for modifying the IGF2 gene of porcine PK15 cells, which further demonstrated a 2.2-fold increase in HDR frequency. In conclusion, our data suggests that Rad52-Cas9 fusion is a good option for enhancing CRISPR/Cas9-mediated HDR, which may be of use in future studies involving precise genome editing.
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