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CRISPR/Cas9 gene editing clarifies the role of CD33 SNP rs12459419 in gemtuzumab ozogamicin-mediated cytotoxicity.
The single-nucleotide polymorphism (SNP) rs12459419 is located at the intron/exon junction of CD33 exon2. When exon2 is skipped by this CD33 SNP, the full-length CD33 (CD33FL) is converted to a short CD33 isoform (CD33D2). Since gemtuzumab ozogamicin (GO) only recognizes CD33FL, the CD33 SNP may affect the clinical efficacy of GO. To elucidate the significance of CD33 SNP on GO reactivity, we leveraged the CRISPR/Cas9 genome-editing system to create OCI-AML3 cell lines with specifically modified CD33 SNPs. Levels of CD33 D2 mRNA were significantly higher in the T/T clone (p < 0.001), but CD33D2 protein was not detectable in any clones. There was no significant difference in CD33FL mRNA expression across edited clones, and CD33FL protein expression was lowest in T/T clones, followed by T/C and C/C. Cytotoxicity assays revealed that the IC50 of GO was significantly lower in T/C and C/C clones than in the T/T clone (p < 0.001). Our study demonstrated a difference in GO-induced cytotoxicity in CD33 SNP-edited clones, clearly indicating that at least one CD33 SNP allele, rs12459419 C, is important for sensitivity to GO.
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Streamlined, single-step non-viral CRISPR-Cas9 knockout strategy enhances gene editing efficiency in primary human chondrocyte populations.
BACKGROUND: CRISPR-Cas9-based genome engineering represents a powerful therapeutic tool for cartilage tissue engineering and for understanding molecular pathways driving cartilage diseases. However, primary chondrocytes are difficult to transfect and rapidly dedifferentiate during monolayer (2D) cell culture, making the lengthy expansion of a single-cell-derived edited clonal population not feasible. For this reason, functional genetics studies focused on cartilage and rheumatic diseases have long been carried out in cellular models that poorly recapitulate the native molecular properties of human cartilaginous tissue (e.g., cell lines, induced pluripotent stem cells). Here, we set out to develop a non-viral CRISPR-Cas9, bulk-gene editing method suitable for chondrocyte populations from different cartilaginous sources. METHODS: We screened electroporation and lipid nanoparticles for ribonucleoprotein (RNP) delivery in primary polydactyly chondrocytes, and optimized RNP reagents assembly. We knocked out RELA (also known as p65), a subunit of the nuclear factor kappa B (NF-κB), in polydactyly chondrocytes and further characterized knockout (KO) cells with RT-qPCR and Western Blot. We tested RELA KO in chondrocytes from diverse cartilaginous sources and characterized their phenotype with RT-qPCR. We examined the chondrogenic potential of wild-type (WT) and KO cell pellets in presence and absence of interleukin-1β (IL-1β). RESULTS: We established electroporation as the optimal transfection technique for chondrocytes enhancing transfection and editing efficiency, while preserving high cell viability. We knocked out RELA with an unprecedented efficiency of ~90%, confirming lower inflammatory pathways activation upon IL-1β stimulation compared to unedited cells. Our protocol could be easily transferred to primary human chondrocytes harvested from osteoarthritis (OA) patients, human FE002 chondroprogenitor cells, bovine chondrocytes, and a human chondrocyte cell line, achieving comparable mean RELA KO editing levels using the same protocol. All KO pellets from primary human chondrocytes retained chondrogenic ability equivalent to WT cells, and additionally displayed enhanced matrix retention under inflamed conditions. CONCLUSIONS: We showcased the applicability of our bulk gene editing method to develop effective autologous and allogeneic off-the-shelf gene therapies strategies and to enable functional genetics studies in human chondrocytes to unravel molecular mechanisms of cartilage diseases.
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The New Frontiers of Gene Therapy and Gene Editing in Inflammatory Diseases.
Inflammatory diseases are conditions characterized by abnormal and often excessive immune responses, leading to tissue and organ inflammation. The complexity of these disorders arises from the intricate interplay of genetic factors and immune responses, which challenges conventional therapeutic approaches. However, the field of genetic manipulation has sparked unprecedented optimism in addressing these complex disorders. This review aims to comprehensively explore the application of gene therapy and gene editing in the context of inflammatory diseases, offering solutions that range from correcting genetic defects to precise immune modulation. These therapies have exhibited remarkable potential in ameliorating symptoms, improving quality of life, and even achieving disease remission. As we delve into recent breakthroughs and therapeutic applications, we illustrate how these advancements offer novel and transformative solutions for conditions that have traditionally eluded conventional treatments. By examining successful case studies and preclinical research, we emphasize the favorable results and substantial transformative impacts that gene-based interventions have demonstrated in patients and animal models of inflammatory diseases such as chronic granulomatous disease, cryopyrin-associated syndromes, and adenosine deaminase 2 deficiency, as well as those of multifactorial origins such as arthropathies (osteoarthritis, rheumatoid arthritis) and inflammatory bowel disease. In conclusion, gene therapy and gene editing offer transformative opportunities to address the underlying causes of inflammatory diseases, ushering in a new era of precision medicine and providing hope for personalized, targeted treatments.
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Manipulation of the Tyrosinase gene permits improved CRISPR/Cas editing and neural imaging in cichlid fish.
Direct tests of gene function have historically been performed in a limited number of model organisms. The CRISPR/Cas system is species-agnostic, offering the ability to manipulate genes in a range of models, enabling insights into evolution, development, and physiology. Astatotilapia burtoni, a cichlid fish from the rivers and shoreline around Lake Tanganyika, has been extensively studied in the laboratory to understand evolution and the neural control of behavior. Here we develop protocols for the creation of CRISPR-edited cichlids and create a broadly useful mutant line. By manipulating the Tyrosinase gene, which is necessary for eumelanin pigment production, we describe a fast and reliable approach to quantify and optimize gene editing efficiency. Tyrosinase mutants also remove a major obstruction to imaging, enabling visualization of subdermal structures and fluorophores in situ. These protocols will facilitate broad application of CRISPR/Cas9 to studies of cichlids as well as other non-traditional model aquatic species.
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Salt and osmotic stress can improve the editing efficiency of CRISPR/Cas9-mediated genome editing system in potato.
CRISPR/Cas9-mediated genome editing technology has been widely used for the study of gene function in crops, but the differences between species have led to widely varying genome editing efficiencies. The present study utilized a potato hairy root genetic transformation system and incorporated a rapid assay with GFP as a screening marker. The results clearly demonstrated that salt and osmotic stress induced by NaCl (10 to 50 mM) and mannitol (50 to 200 mM) treatments significantly increased the positive rates of genetic transformation mediated by A. rhizogenes and the editing efficiency of the CRISPR/Cas9-mediated genome editing system in potato. However, it was observed that the regeneration of potato roots was partially inhibited as a result. The analysis of CRISPR/Cas9-mediated mutation types revealed that chimeras accounted for the largest proportion, ranging from 62.50% to 100%. Moreover, the application of salt and osmotic stress resulted in an increased probability of null mutations in potato. Notably, the highest rate of null mutations, reaching 37.5%, was observed at a NaCl concentration of 10 mM. Three potential off-target sites were sequenced and no off-targeting was found. In conclusion, the application of appropriate salt and osmotic stress significantly improved the editing efficiency of the CRISPR/Cas9-mediated genome editing system in potato, with no observed off-target effects. However, there was a trade-off as the regeneration of potato roots was partially inhibited. Overall, these findings present a new and convenient approach to enhance the genome editing efficiency of the CRISPR/Cas9-mediated gene editing system in potato.
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Compact Cas12f enables genome editing in avian cells.
Precise genome editing in avian species has been constrained by the low delivery efficiency of conventional CRISPR nucleases, such as Cas9 and Cas12a, due to their large molecular sizes. Cas12f (also known as Cas14), a compact CRISPR nuclease, has emerged as a potential genome editing system with enhanced delivery efficiency in mammalian systems. However, its effectiveness in avian systems has not been previously validated. Here, Cas12f showed notable transfection efficiency and intracellular expression in chicken Leghorn male hepatoma (LMH) cells and primordial germ cells (PGCs), with no detectable cytotoxicity. Next-generation sequencing (NGS) revealed that Cas12f achieved locus-dependent on-target editing efficiencies, reaching up to 40% at specific loci in LMH cells. Cas12f consistently generated a deletion-dominant indel profile with minimal insertions, distinct from Cas9-mediated patterns. Off-target analysis using Sanger sequencing and Inference of CRISPR Edits (ICE) revealed a few predicted off-target candidates and no detectable off-target mutations above the detection threshold. Consistent with this observation, cross-species in silico analysis showed only a modest increase in predicted Cas12f off-target proportions with increasing genome size. These findings show that Cas12f is a compact genome editing tool in avian cells, serving as a basis for further improvement in genetic engineering and biotechnological research.
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CRISPR/Cas gene-editing technology and its advances in dentistry.
A recent discovery of revolutionary Clustered regularly interspaced palindromic repeats (CRISPR) is a gene-editing tool that provides a type of adaptive immunity in prokaryotic organisms, which is currently used as a revolutionizing tool in biomedical research. It has a mechanism of correcting genome errors, turning on/off genes in cells and organisms. Most importantly playing a crucial function in bacterial defence by identifying and destroying Deoxyribonucleic acid (DNA) segments during bacteriophage invasions since the CRISPR-associated protein 9 (Cas9) enzyme recognizes and cleaves invasive DNA sequences complementary to CRISPR. Therefore, researchers employ this biological device to manipulate the genes to develop new therapies to combat systemic diseases. Currently, the most significant advance at the laboratory level is the generation of cell and animal models, functional genomic screens, live images of the cell genome, and defective DNA repairs to find the cure for genetic disorders. Even though this technology has enormous biomedical applications in various sectors, this review will summarize CRISPR/Cas emphasizing both the therapeutic and diagnostic mechanisms developed in the field of dentistry and the promising attempts to transfer this technology to clinical application. Finally, future developments are also described, which proposes to use CRISPR/Cas systems for prospective clinical dentistry applications.
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Prime Editing Promises Greater Precision, Safety.
Cancer researchers are heralding the arrival of prime editing, an ultra-precise twist on CRISPR genome engineering, as a powerful new tool for disease modeling, target validation, and clinical applications.
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Electroporation Delivery of Cas9 sgRNA Ribonucleoprotein-Mediated Genome Editing in Sheep IVF Zygotes.
The utilization of electroporation for delivering CRISPR/Cas9 system components has enabled efficient gene editing in mammalian zygotes, facilitating the development of genome-edited animals. In this study, our research focused on targeting the ACTG1 and MSTN genes in sheep, revealing a threshold phenomenon in electroporation with a voltage tolerance in sheep in vitro fertilization (IVF) zygotes. Various poring voltages near 40 V and pulse durations were examined for electroporating sheep zygotes. The study concluded that stronger electric fields required shorter pulse durations to achieve the optimal conditions for high gene mutation rates and reasonable blastocyst development. This investigation also assessed the quality of Cas9/sgRNA ribonucleoprotein complexes (Cas9 RNPs) and their influence on genome editing efficiency in sheep early embryos. It was highlighted that pre-complexation of Cas9 proteins with single-guide RNA (sgRNA) before electroporation was essential for achieving a high mutation rate. The use of suitable electroporation parameters for sheep IVF zygotes led to significantly high mutation rates and heterozygote ratios. By delivering Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) to zygotes through electroporation, targeting the MSTN (Myostatin) gene, a knock-in efficiency of 26% was achieved. The successful generation of MSTN-modified lambs was demonstrated by delivering Cas9 RNPs into IVF zygotes via electroporation.
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What to Expect When Expecting CRISPR Baby Number Four.
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