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CRISPR/Cas9 editing in conditionally immortalized HoxB8 cells for studying gene regulation in mouse dendritic cells.
HoxB8 multipotent progenitors (MPP) are obtained by expression of the estrogen receptor hormone binding domain (ERHBD) HoxB8 fusion gene in mouse BM cells. HoxB8 MPP generate (i) the full complement of DC subsets (cDC1, cDC2, and pDC) in vitro and in vivo and (ii) allow CRISPR/Cas9-mediated gene editing, for example, generating homozygous deletions in cis-acting DNA elements at high precision, and (iii) efficient gene repression by dCas9-KRAB for studying gene regulation in DC differentiation.
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A CRISPR-Cas9 system for knock-out and knock-in of high molecular weight DNA enables module-swapping of the pikromycin synthase in its native host.
BACKGROUND: Engineers seeking to generate natural product analogs through altering modular polyketide synthases (PKSs) face significant challenges when genomically editing large stretches of DNA. RESULTS: We describe a CRISPR-Cas9 system that was employed to reprogram the PKS in Streptomyces venezuelae ATCC 15439 that helps biosynthesize the macrolide antibiotic pikromycin. We first demonstrate its precise editing ability by generating strains that lack megasynthase genes pikAI-pikAIV or the entire pikromycin biosynthetic gene cluster but produce pikromycin upon complementation. We then employ it to replace 4.4-kb modules in the pikromycin synthase with those of other synthases to yield two new macrolide antibiotics with activities similar to pikromycin. CONCLUSION: Our gene-editing tool has enabled the efficient replacement of extensive and repetitive DNA regions within streptomycetes.
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Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9.
The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5'-NGG-3' protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutations in zebrafish. Bioinformatics analysis of these new Cas targets suggests that the number of available target sites in the zebrafish genome can be greatly expanded. Collectively, the expanded target repertoire of Cas9 in zebrafish should further facilitate the utility of this organism for genetic studies of vertebrate biology.
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Enhanced genome editing in rice using single transcript unit CRISPR-LbCpf1 systems.
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From "Simulation" to "Mirror": Gene editing and humanization redefines the next-generation precision oncology animal model.
Patient-derived xenograft (PDX) models, although conventionally used in oncology, exhibit critical limitations: they frequently lose patient-specific genetic mutations and lack the human leukocyte antigen (HLA) diversity essential for immune recognition, and fail to recapitulate the human tumor microenvironment (TME). These deficiencies contribute to immunotherapy prediction failure rates exceeding 80% in clinical translation. To address these gaps, we propose a Tumor Model 2.0 framework. This framework integrates multi-omics data (whole-genome, transcriptomic, and proteomic) with precision genome editing technologies (CRISPR-Cas9 and Prime Editing) to reconstruct patient-specific mutations across multiple biological layers. Employing an organoid-animal coupling platform with stepwise immune system construction and microenvironment remodeling-subsequently validated in large animals-the framework enables the creation of programmable, patient-specific digital twins. These high-fidelity models support personalized N-of-1 clinical trials, bridging the gap between preclinical research and clinical precision oncology.
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Rational Design of ssODN to Correct Mutations by Gene Editing.
Gene editing allows to make a variety of targeted changes in genome, which can potentially be used to treat hereditary human diseases. Despite numerous studies in this area, effectiveness of gene editing methods for correcting mutations is still low, so these methods are not allowed in routine practice. It has been shown that rational design of genome editing components can significantly increase efficiency of mutation correction. In this work, we propose design of single-stranded oligodeoxyribonucleotides (ssODNs) to increase efficiency of gene editing. Using a model system to repair knocked out EGFP that is integrated into the genome of HEK293T cell culture, we have shown that only a small part of ssODN (about 20 nucleotides: from the 15th nucleotide at 3'-end to the 4th nucleotide at 5'-end), a donor molecule for repairing double-stranded DNA breaks, is integrated into the site of the break. Based on the obtained data, it is possible to rationally approach the design of ssODNs to correct mutations using CRISPR-Cas9 method for the development of gene therapy for hereditary human diseases.
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Emerging applications of gene edited T cells for the treatment of leukemia.
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Genome editing for disease locus dissection.
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Construction of a CRISPR/nCas9-assisted genome editing system for exopolysaccharide biosynthesis in Streptococcus thermophilus.
Streptococcus thermophilus is an economically prominent starter for common dairy products due to its potential health and nutritional benefits. However, lack of precise genetic manipulation approaches has greatly hampered the industrial application of S. thermophilus.. Herein, we developed an efficient genome editing toolbox (pKLH353) based on CRISPR/nCas9 (Cas9 nickase) in S. thermophilus to seamlessly edit single or multiple genes. A native constitutive promoter library was used to optimize the nCas9 and sgRNA expression with gene deletion efficiencies of 14-60%. The epsA, epsB and epsE were identified as key genes affecting exopolysaccharide (EPS) biosynthesis in S. thermophilus S-3 using the CRISPR/nCas9 toolbox. Moreover, compared to the wild-type, knockout of epsC, epsE or epsG led to a decrease of EPS titer with reducing in its molecular weight (>2.5-fold) and intrinsic viscosity (>19.8-fold). The ratio of monosaccharide composition of the mutants has also changed, suggesting that these eps genes are involved in the chain length synthesis and repeat unit assembly. Taken together, this CRISPR/nCas9 system can serve as a basic toolkit for precise genetic engineering of S. thermophilus and facilitate strain engineering to produce bio-based products.
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AI Revolutionizing Cell and Genetic Engineering: Innovations and Applications.
The integration of Artificial Intelligence (AI) into the realms of cell and genetic engineering has ushered in a transformative era of innovation and discovery. This chapter explores the myriad ways in which AI technologies are revolutionizing these fields, from data analysis and drug discovery to genomic sequencing and gene editing. AI algorithms are adept at analyzing vast datasets, uncovering intricate patterns, and predicting biological phenomena with unparalleled accuracy. In drug discovery, AI-driven platforms expedite the identification of potential therapeutics by simulating molecular interactions and predicting their efficacy. Genomic sequencing efforts benefit from AI's ability to interpret genetic variations and their implications for health and disease. Furthermore, AI-guided gene editing techniques, such as CRISPR-Cas9, enable precise and targeted modifications of the genome. Beyond the laboratory, AI facilitates personalized medicine by analyzing genetic data to tailor treatments to individual patients. This chapter underscores the pivotal role of AI in advancing cell and genetic engineering, promising a future of unprecedented scientific breakthroughs and personalized healthcare solutions.
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