FTH1 overexpression using a dCasRx translation enhancement system protects the kidney from calcium oxalate crystal-induced injury.

The engineered clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system is currently widely applied in genetic editing and transcriptional regulation. The catalytically inactivated CasRx (dCasRx) has the ability to selectively focus on the mRNA coding region without disrupting transcription and translation, opening up new avenues for research on RNA modification and protein translation control. This research utilized dCasRx to create a translation-enhancement system for mammals called dCasRx-eIF4GI, which combined eukaryotic translation initiation factor 4G (eIF4GI) to boost translation levels of the target gene by recruiting ribosomes, without affecting mRNA levels, ultimately increasing translation levels of different endogenous proteins. Due to the small size of dCasRx, the dCasRx-eIF4GI translation enhancement system was integrated into a single viral vector, thus optimizing the delivery and transfection efficiency in subsequent applications. Previous studies reported that ferroptosis, mediated by calcium oxalate (CaOx) crystals, significantly promotes stone formation. In order to further validate its developmental potential, it was applied to a kidney stone model in vitro and in vivo. The manipulation of the ferroptosis regulatory gene FTH1 through single-guide RNA (sgRNA) resulted in a notable increase in FTH1 protein levels without affecting its mRNA levels. This ultimately prevented intracellular ferroptosis and protected against cell damage and renal impairment caused by CaOx crystals. Taken together, this study preliminarily validated the effectiveness and application prospects of the dCasRx-eIF4GI translation enhancement system in mammalian cell-based disease models, providing novel insights and a universal tool platform for protein translation research and future therapeutic approaches for nephrolithiasis.

Emerging strategies for treating autoimmune disease with genetically modified dendritic cells.

Gene editing of living cells has become a crucial tool in medical research, enabling scientists to address fundamental biological questions and develop novel strategies for disease treatment. This technology has particularly revolutionized adoptive transfer cell therapy products, leading to significant advancements in tumor treatment and offering promising outcomes in managing transplant rejection, autoimmune disorders, and inflammatory diseases. While recent clinical trials have demonstrated the safety of tolerogenic dendritic cell (TolDC) immunotherapy, concerns remain regarding its effectiveness. This review aims to discuss the application of gene editing techniques to enhance the tolerance function of dendritic cells (DCs), with a particular focus on preclinical strategies that are currently being investigated to optimize the tolerogenic phenotype and function of DCs. We explore potential approaches for in vitro generation of TolDCs and provide an overview of emerging strategies for modifying DCs. Additionally, we highlight the primary challenges hindering the clinical adoption of TolDC therapeutics and propose future research directions in this field.

A new dawn for the study of cell type evolution.

Animal evolution is influenced by the emergence of new cell types, yet our understanding of this process remains elusive. This prompts the need for a broader exploration across diverse research organisms, facilitated by recent breakthroughs, such as gene editing tools and single-cell genomics. Essential to our understanding of cell type evolution is the accurate identification of homologous cells. We delve into the significance of considering developmental ontogeny and potential pitfalls when drawing conclusions about cell type homology. Additionally, we highlight recent discoveries in the study of cell type evolution through the application of single-cell transcriptomics and pinpoint areas ripe for further exploration.

Enhancing genome editing in hPSCs through dual inhibition of DNA damage response and repair pathways.

Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived hPSCs. Unlike Cas9-mediated knock-in, cytosine base editor and prime editor achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by cytosine base editor or prime editor additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of p53DD, a dominant negative p53, and three UNG inhibitor, engineered to specifically diminish base excision repair, improves cytosine base editor efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional prime editor system also significantly enhances prime editor efficiency in hPSCs. Thus, combined inhibition of the distinct cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.

CRISPR-based gene expression platform for precise regulation of bladder cancer.

The development of compact CRISPR systems has facilitated delivery but has concurrently reduced gene editing efficiency, thereby limiting the further utilization of CRISPR systems. Enhancing the efficiency of CRISPR systems poses a challenging task and holds significant implications for the advancement of biotechnology. In our work, we report a synthetic dual-antibody system that can stably exist in the intracellular environment, specifically inhibiting the functions of NF-κB and ß-catenin. This not only elevates the transgenic expression of the CRISPR system by suppressing the innate immune response within cells to enhance the gene editing efficiency but also demonstrates a notable tumor inhibitory effect. Based on the specific output expression regulation of CRISPR-CasΦ, we constructed a CRISPR-based gene expression platform, which includes sensor modules for detecting intracellular ß-catenin and NF-κB, as well as an SDA module to enhance overall efficiency. In vitro experiments revealed that the CRISPR-based gene expression platform exhibited superior CDK5 expression inhibition efficiency and specific cytotoxicity towards tumor cells. In vitro experiments, we found that CRISPR-based gene expression platforms can selectively kill bladder cancer cells through T cell-mediated cytotoxicity. Our design holds significant assistant potential of transgene therapy and may offer the capability to treat other diseases requiring transgene therapy.

Prime editing-mediated correction of the leptin receptor in muscle cells of db/db mice.

Genetic diseases can be caused by monogenic diseases, which result from a single gene mutation in the DNA sequence. Many innovative approaches have been developed to cure monogenic genetic diseases, namely by genome editing. A specific type of genomic editing, prime editing, has the potential advantage to edit the human genome without requiring double-strand breaks or donor DNA templates for editing. Additionally, prime editing does not require a precisely positioned protospacer adjacent motif (PAM) sequence, which offers flexible target and more precise genomic editing. Here we detail a novel construction of a prime editing extended guide RNA (pegRNA) to target mutated leptin receptors in B6.BKS(D)-Leprdb/J mice (db/db mice). The pegRNA was then injected into the flexor digitorum brevis (FDB) muscle of db/db mice to demonstrate in vivo efficacy, which resulted in pegRNA mediated base transversion at endogenous base transversion. Genomic DNA sequencing confirmed that prime editing could correct the mutation of leptin receptor gene in db/db mice. Furthermore, prime editing treated skeletal muscle exhibited enhanced leptin receptor signals. Thus, the current study showed in vivo efficacy of prime editing to correct mutant protein and rescue the physiology associated with functional protein.

Fusion of histone variants to Cas9 suppresses non-homologous end joining.

As a versatile genome editing tool, the CRISPR-Cas9 system induces DNA double-strand breaks at targeted sites to activate mainly two DNA repair pathways: HDR which allows precise editing via recombination with a homologous template DNA, and NHEJ which connects two ends of the broken DNA, which is often accompanied by random insertions and deletions. Therefore, how to enhance HDR while suppressing NHEJ is a key to successful applications that require precise genome editing. Histones are small proteins with a lot of basic amino acids that generate electrostatic affinity to DNA. Since H2A.X is involved in DNA repair processes, we fused H2A.X to Cas9 and found that this fusion protein could improve the HDR/NHEJ ratio by suppressing NHEJ. As various post-translational modifications of H2A.X play roles in the regulation of DNA repair, we also fused H2A.X mimicry variants to replicate these post-translational modifications including phosphorylation, methylation, and acetylation. However, none of them were effective to improve the HDR/NHEJ ratio. We further fused other histone variants to Cas9 and found that H2A.1 suppressed NHEJ better than H2A.X. Thus, the fusion of histone variants to Cas9 is a promising option to enhance precise genome editing.

Establishment and characterization of Lesch-Nyhan syndrome rabbit model.

Lesch-Nyhan syndrome (LNS) is a congenital defect disease that results in defective purine metabolism. It is caused by pathogenic variants of the HPRT gene. Its clinical symptoms mainly include high uric acid levels, gout, and kidney stones and damage. The mechanism of LNS has not been fully elucidated, and no cure exists. Animal models have always played an important role in exploring causative mechanisms and new therapies. This study combined CRISPR/Cas9 and microinjection to knock out the HPRT gene to create an LNS rabbit model. A sgRNA targeting exon 3 of HPRT gene was designed. Subsequently, Cas9 mRNA and sgRNA were injected into rabbit zygotes, and injected embryos were transferred to the uterus. The genotype and phenotype of rabbits were analyzed after birth. Four infant rabbits (named R1, R2, R3 and R4), which showed varying levels of gene modification, were born. The gene-editing efficiency was 100%. No wild-type sequences at the target HPRT gene were detected in R4 rabbit. Next, 6-thioguanine drug testing confirmed that HPRT enzymatic activity was deficient in R4 infant rabbit. HE staining revealed kidney abnormalities in all infant rabbits. Overall, an sgRNA capable of knocking out the HPRT gene in rabbits was successfully designed, and HPRT gene-modified rabbits were successfully constructed by using CRISPR/Cas9 technology and microinjection. This study provides a new nonrodent animal model for studying LNS syndrome.

Programmable RNA targeting with CRISPR-Cas13.

The RNA-targeting CRISPR-Cas13 system has enabled precise engineering of endogenous RNAs, significantly advancing our understanding of RNA regulation and the development of RNA-based diagnostic and therapeutic applications. This review aims to provide a summary of Cas13-based RNA targeting tools and applications, discuss limitations and challenges of existing tools and suggest potential directions for further development of the RNA targeting system.

An antifouling membrane-fusogenic liposome for effective intracellular delivery in vivo.

The membrane-fusion-based internalization without lysosomal entrapment is advantageous for intracellular delivery over endocytosis. However, protein corona formed on the membrane-fusogenic liposome surface converts its membrane-fusion performance to lysosome-dependent endocytosis, causing poorer delivery efficiency in biological conditions. Herein, we develop an antifouling membrane-fusogenic liposome for effective intracellular delivery in vivo. Leveraging specific lipid composition at an optimized ratio, such antifouling membrane-fusogenic liposome facilitates fusion capacity even in protein-rich conditions, attributed to the copious zwitterionic phosphorylcholine groups for protein-adsorption resistance. Consequently, the antifouling membrane-fusogenic liposome demonstrates robust membrane-fusion-mediated delivery in the medium with up to 38% fetal bovine serum, outclassing two traditional membrane-fusogenic liposomes effective at 4% and 6% concentrations. When injected into mice, antifouling membrane-fusogenic liposomes can keep their membrane-fusion-transportation behaviors, thereby achieving efficient luciferase transfection and enhancing gene-editing-mediated viral inhibition. This study provides a promising tool for effective intracellular delivery under complex physiological environments, enlightening future nanomedicine design.

Exonuclease editor promotes precision of gene editing in mammalian cells.

BACKGROUND: Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be challenging. RESULTS: Here, we have developed a human exonuclease 1-based genome-editing tool, referred to as exonuclease editor. When compared to Cas9, the exonuclease editor gave rise to increased HDR efficiency, reduced NHEJ repair frequency, and significantly elevated HDR/indel ratio. Robust gene editing precision of exonuclease editor was even superior to the fusion of Cas9 with E1B or DN1S, two previously reported precision-enhancing domains. Notably, exonuclease editor inhibited NHEJ at double strand breaks locally rather than globally, reducing indel frequency without compromising genome integrity. The replacement of Cas9 with single-strand DNA break-creating Cas9 nickase further increased the HDR/indel ratio by 453-fold than the original Cas9. In addition, exonuclease editor resulted in high microhomology-mediated end joining efficiency, allowing accurate and flexible deletion of targeted sequences with extended lengths with the aid of paired sgRNAs. Exonuclease editor was further used for correction of DMD patient-derived induced pluripotent stem cells, where 30.0% of colonies were repaired by HDR versus 11.1% in the control. CONCLUSIONS: Therefore, the exonuclease editor system provides a versatile and safe genome editing tool with high precision and holds promise for therapeutic gene correction.

KCTD Proteins Have Redundant Functions in Controlling Cellular Growth.

We explored the functional redundancy of three structurally related KCTD (Potassium Channel Tetramerization Domain) proteins, KCTD2, KCTD5, and KCTD17, by progressively knocking them out in HEK 293 cells using CRISPR/Cas9 genome editing. After validating the knockout, we assessed the effects of progressive knockout on cell growth and gene expression. We noted that the progressive effects of knockout of KCTD isoforms on cell growth were most pervasive when all three isoforms were deleted, suggesting some functions were conserved between them. This was also reflected in progressive changes in gene expression. Our previous work indicated that Gß1 was involved in the transcriptional control of gene expression, so we compared the gene expression patterns between GNB1 and KCTD KO. Knockout of GNB1 led to numerous changes in the expression levels of other G protein subunit genes, while knockout of KCTD isoforms had the opposite effect, presumably because of their role in regulating levels of Gß1. Our work demonstrates a unique relationship between KCTD proteins and Gß1 and a global role for this subfamily of KCTD proteins in maintaining the ability of cells to survive and proliferate.

CRISPR-Cas9 delivery strategies and applications: Review and update.

Nowadays, a significant part of the investigations carried out in the medical field belong to cancer treatment. Generally, conventional cancer treatments, including chemotherapy, radiotherapy, and surgery, which have been used for a long time, are not sufficient, especially in malignant cancers. Because genetic mutations cause cancers, researchers are trying to treat these diseases using genetic engineering tools. One of them is clustered regularly interspaced short palindromic repeats (CRISPR), a powerful tool in genetic engineering in the last decade. CRISPR, which forms the CRISPR-Cas structure with its endonuclease protein, Cas, is known as a part of the immune system (adaptive immunity) in bacteria and archaea. Among the types of Cas proteins, Cas9 endonuclease has been used in many scientific studies due to its high accuracy and efficiency. This review reviews the CRISPR system, focusing on the history, classification, delivery methods, applications, new generations, and challenges of CRISPR-Cas9 technology.

Development of a New Model System to Study Long-Distance Interactions Supported by Architectural Proteins.

Chromatin architecture is critical for the temporal and tissue-specific activation of genes that determine eukaryotic development. The functional interaction between enhancers and promoters is controlled by insulators and tethering elements that support specific long-distance interactions. However, the mechanisms of the formation and maintenance of long-range interactions between genome regulatory elements remain poorly understood, primarily due to the lack of convenient model systems. Drosophila became the first model organism in which architectural proteins that determine the activity of insulators were described. In Drosophila, one of the best-studied DNA-binding architectural proteins, Su(Hw), forms a complex with Mod(mdg4)-67.2 and CP190 proteins. Using a combination of CRISPR/Cas9 genome editing and attP-dependent integration technologies, we created a model system in which the promoters and enhancers of two reporter genes are separated by 28 kb. In this case, enhancers effectively stimulate reporter gene promoters in cis and trans only in the presence of artificial Su(Hw) binding sites (SBS), in both constructs. The expression of the mutant Su(Hw) protein, which cannot interact with CP190, and the mutation inactivating Mod(mdg4)-67.2, lead to the complete loss or significant weakening of enhancer-promoter interactions, respectively. The results indicate that the new model system effectively identifies the role of individual subunits of architectural protein complexes in forming and maintaining specific long-distance interactions in the D. melanogaster model.

CRISPR-Cas9 genome and long non-coding RNAs as a novel diagnostic index for prostate cancer therapy via liposomal-coated compounds.

CRISPR/Cas9 is a recently discovered genomic editing technique that altered scientist's sight in studying genes function. Cas9 is controlled via guide (g) RNAs, which match the DNA targeted in cleavage to modify the respective gene. The development in prostate cancer (PC) modeling directed not only to novel resources for recognizing the signaling pathways overriding prostate cell carcinoma, but it has also created a vast reservoir for complementary tools to examine therapies counteracting this type of cancer. Various cultured somatic rat models for prostate cancer have been developed that nearly mimic human prostate cancer. Nano-medicine can passively target cancer cells via increasing bioavailability and conjugation via specific legend, contributing to reduced systemic side-effects and increased efficacy. This article highlights liposomal loaded Nano-medicine as a potential treatment for prostate cancer and clarifies the CRISPR/Cas9 variation accompanied with prostate cancer. PC is induced experimentally in western rat model via ethinyl estradiol for 4 weeks and SC. dose of 3, 2'- dimethyl-4-aminobiphenyl estradiol (DAE) (50mg/kg) followed by treatment via targeted liposomal-coated compounds such as liposomal dexamethasone (DXM), liposomal doxorubicin (DOX) and liposomal Turmeric (TUR) (3mg/kg IP) for four weeks in a comparative study to their non-targeted analogue dexamethasone, doxorubicin and Turmeric. 3, 2'- dimethyl-4-aminobiphenylestradiol elicit prostate cancer in western rats within 5 months. Simultaneous supplementations with these liposomal compounds influence on prostate cancer; tumor markers were investigated via prostate-specific antigen (PSA), Nitric oxide (NOX) and CRISPR/Cas9 gene editing. Several long non-coding RNAs were reported to be deregulated in prostate cell carcinoma, including MALAT1. On the other hand, gene expression of apoptotic biomarkers focal adhesion kinase (AKT-1), phosphatidylinistol kinase (PI3K) and glycogen synthase kinase-3 (GSK-3) was also investigated and further confirming these results via histopathological examination. Liposomal loaded dexamethasone; doxorubicin and Turmeric can be considered as promising therapeutic agents for prostate cancer via modulating CRISPR/Cas9 gene editing and long non coding gene MALAT1.

Conditional knockdown of OsMLH1 to improve plant prime editing systems without disturbing fertility in rice.

BACKGROUND: High-efficiency prime editing (PE) is desirable for precise genome manipulation. The activity of mammalian PE systems can be largely improved by inhibiting DNA mismatch repair by coexpressing a dominant-negative variant of MLH1. However, this strategy has not been widely used for PE optimization in plants, possibly because of its less conspicuous effects and inconsistent performance at different sites. RESULTS: We show that direct RNAi knockdown of OsMLH1 in an ePE5c system increases the efficiency of our most recently updated PE tool by 1.30- to 2.11-fold in stably transformed rice cells, resulting in as many as 85.42% homozygous mutants in the T0 generation. The high specificity of ePE5c is revealed by whole-genome sequencing. To overcome the partial sterility induced by OsMLH1 knockdown of ePE5c, a conditional excision system is introduced to remove the RNAi module by Cre-mediated site-specific recombination. Using a simple approach of enriching excision events, we generate 100% RNAi module-free plants in the T0 generation. The increase in efficiency due to OsMLH1 knockdown is maintained in the excised plants, whose fertility is not impaired. CONCLUSIONS: This study provides a safe and reliable plant PE optimization strategy for improving editing efficiency without disturbing plant development via transient MMR inhibition with an excisable RNAi module of MLH1.

Small extracellular vesicles (sEVs)-based gene delivery platform for cell-specific CRISPR/Cas9 genome editing.

Small extracellular vesicles (sEVs) are naturally occurring vesicles that have the potential to be manipulated to become promising drug delivery vehicles for on-demand in vitro and in vivo gene editing. Here, we developed the modular safeEXO platform, a prototype sEV delivery vehicle that is mostly devoid of endogenous RNA and can efficaciously deliver RNA and ribonucleoprotein (RNP) complexes to their intended intracellular targets manifested by downstream biologic activity. We also successfully engineered producer cells to produce safeEXO vehicles that contain endogenous Cas9 (safeEXO-CAS) to effectively deliver efficient ribonucleoprotein (RNP)-mediated CRISPR genome editing machinery to organs or diseased cells in vitro and in vivo. We confirmed that safeEXO-CAS sEVs could co-deliver ssDNA, sgRNA and siRNA, and efficaciously mediate gene insertion in a dose-dependent manner. We demonstrated the potential to target safeEXO-CAS sEVs by engineering sEVs to express a tissue-specific moiety, integrin alpha-6 (safeEXO-CAS-ITGA6), which increased their uptake to lung epithelial cells in vitro and in vivo. We tested the ability of safeEXO-CAS-ITGA6 loaded with EMX1 sgRNAs to induce lung-targeted editing in mice, which demonstrated significant gene editing in the lungs with no signs of morbidity or detectable changes in immune cell populations. Our results demonstrate that our modular safeEXO platform represents a targetable, safe, and efficacious vehicle to deliver nucleic acid-based therapeutics that successfully reach their intracellular targets. Furthermore, safeEXO producer cells can be genetically manipulated to produce safeEXO vehicles containing CRISPR machinery for more efficient RNP-mediated genome editing. This platform has the potential to improve current therapies and increase the landscape of treatment for various human diseases using RNAi and CRISPR approaches.

ß-Thalassemia gene editing therapy: Advancements and difficulties.

ß-Thalassemia is the world's number 1 single-gene genetic disorder and is characterized by suppressed or impaired production of ß-pearl protein chains. This results in intramedullary destruction and premature lysis of red blood cells in peripheral blood. Among them, patients with transfusion-dependent ß-thalassemia face the problem of long-term transfusion and iron chelation therapy, which leads to clinical complications and great economic stress. As gene editing technology improves, we are seeing the dawn of a cure for the disease, with its reduction of ineffective erythropoiesis and effective prolongation of survival in critically ill patients. Here, we provide an overview of ß-thalassemia distribution and pathophysiology. In addition, we focus on gene therapy and gene editing advances. Nucleic acid endonuclease tools currently available for gene editing fall into 3 categories: zinc finger nucleases, transcription activator-like effector nucleases, and regularly interspaced short palindromic repeats (CRISPR-Cas9) nucleases. This paper reviews the exploratory applications and exploration of emerging therapeutic tools based on 3 classes of nucleic acid endonucleases in the treatment of ß-thalassemia diseases.

Genome editing: An insight into disease resistance, production efficiency, and biomedical applications in livestock.

One of the primary concerns for the survival of the human species is the growing demand for food brought on by an increasing global population. New developments in genome-editing technology present promising opportunities for the growth of wholesome and prolific farm animals. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. Genome editing entails modifying genetic material by removing, adding, or manipulating particular DNA sequences from a particular locus in a way that does not happen naturally. The three primary genome editors are CRISPR/Cas 9, TALENs, and ZFNs. Each of these enzymes is capable of precisely severing nuclear DNA at a predetermined location. One of the most effective inventions is base editing, which enables single base conversions without the requirement for a DNA double-strand break (DSB). As reliable methods for precise genome editing in studies involving animals, cytosine and adenine base editing are now well-established. Effective zygote editing with both cytosine and adenine base editors (ABE) has resulted in the production of animal models. Both base editors produced comparable outcomes for the precise editing of point mutations in somatic cells, advancing the field of gene therapy. This review focused on the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of ZFNs, TALENs, and CRISPR/Cas9 base editors, and prime editing in diverse lab and farm animals. Additionally, we address the methodologies that can be used for gene regulation, base editing, and epigenetic alterations, as well as the significance of genome editing in animal models to better reflect real disease. We also look at methods designed to increase the effectiveness and precision of gene editing tools. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. This review is an overview of the existing knowledge of the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of zinc finger nucleases (ZFNs), transcription-activator-like endonucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas 9), base editors and prime editing in diverse lab and farm animals, which will offer better and healthier products for the entire human race.