Gene Editing

Gene drives may be capable of addressing ecological problems by altering entire populations of wild organisms, but their use has remained largely theoretical due to technical constraints. Here we consider the potential for RNA-guided gene drives based on the CRISPR nuclease Cas9 to serve as a general method for spreading altered traits through wild populations over many generations. We detail likely capabilities, discuss limitations, and provide novel precautionary strategies to control the spread of gene drives and reverse genomic changes. The ability to edit populations of sexual species would offer substantial benefits to humanity and the environment. For example, RNA-guided gene drives could potentially prevent the spread of disease, support agriculture by reversing pesticide and herbicide resistance in insects and weeds, and control damaging invasive species. However, the possibility of unwanted ecological effects and near-certainty of spread across political borders demand careful assessment of each potential application. We call for thoughtful, inclusive, and well-informed public discussions to explore the responsible use of this currently theoretical technology.

Wheat (Triticum aestivum L.) is the third most important cereal crop of Nepal in terms of area and production. Spot blotch caused by Bipolaris sorokiniana Sacc. is one of the major diseases reducing the productivity and quality of wheat in Nepal, which is favored by high temperature and relative humidity. The experiment was conducted at research farm of Rampur Campus, Khairahani, Chitwan from 18 th Nov 2021 to 29 th Mar 2022, to identify the genotypes resistance against spot blotch. It was laid out in Randomized complete block design (RCBD) with twelve genotypes as treatments and three replications. Double digit scoring method was used to assess the disease five times from 90 DAS at the interval of 7 days and Area under disease progress curve (AUDPC) was calculated. Statistically, significant difference was obtained in all the observed traits except for plant height, spike weight, harvest index, and straw yield. The highest AUDPC value was recorded in RR-21 (2074) yielding 4.0 tonha -1 whereas the lowest AUDPC value was recorded in NL 1437 (781) yielding 6.1 tonha -1 , suggesting it to be more resistant than other genotypes. NL 1447 was the highest yielder (6.5tonha - 1 ) with a 985.185 mean AUDPC value followed by NL1437 (6.1 tonha -1 ) while RR-21 was the lowest yielder (4.0 tonha -1 ). The lowest apparent infection rate was detected for BL 4951 (0.018). Correlation studies indicated that total grains and grain weight per spike as well as the grain yield were negatively and significantly correlated with AUDPC.

The CRISPR/Cas9 system has been employed to efficiently edit the genomes of diverse model organisms. CRISPR-mediated mouse genome editing is typically accomplished by microinjection of Cas9 DNA/RNA and single-guide RNA into zygotes to generate modified animals in one step. However, microinjection is a technically demanding, labor-intensive, and costly procedure with poor embryo viability. Here, we describe a simple and economic electroporation-based strategy to deliver Cas9/sgRNA ribonucleoproteins (RNPs) into mouse zygotes with a 100% efficiency for in vivo genome editing. Our methodology, designated as CRISPR RNP Electroporation of Zygotes (CRISPR-EZ), enables highly efficient and high-throughput genome editing in vivo, with a significant improvement in embryo viability compared to microinjection. Using CRISPR-EZ, we generated a variety of editing schemes in mouse embryos, including indel (insertion/deletion) mutations, point mutations, large deletions, and small insertions. In a ...

Tooth loss is a prevalent health issue that traditional solutions address through either prosthodontic restorations or dental implants. Laboratory-produced bioengineered teeth are no longer a scientific speculation, as regenerative medicine continues to make innovative progress. The critical advancement within this research area relies on USAG-1 (Uterine Sensitization Associated Gene-1) gene suppression which blocks tooth development. Scientists have shown that USAG-1 inhibition leads to supernumerary tooth growth through recent studies thereby creating a modern dental therapeutic option. The investigative analysis reviews existing knowledge about lab-grown teeth to explore USAG-1 regulation of dental tissue regeneration together with potential treatments despite implementation barriers.

Basal forebrain cholinergic neurons (BFCNs) are believed to be one of the first cell types to be affected in all forms of AD, and their dysfunction is clinically correlated with impaired short-term memory formation and retrieval. We present an optimized in vitro protocol to generate human BFCNs from iPSCs, using cell lines from presenilin 2 (PSEN2) mutation carriers and controls. As expected, cell lines harboring the PSEN2 (N141I) mutation displayed an increase in the Aβ42/40 in iPSC-derived BFCNs. Neurons derived from PSEN2 (N141I) lines generated fewer maximum number of spikes in response to a square depolarizing current injection. The height of the first action potential at rheobase current injection was also significantly decreased in PSEN2 (N141I) BFCNs. CRISPR/Cas9 correction of the PSEN2 point mutation abolished the electrophysiological deficit, restoring both the maximal number of spikes and spike height to the levels recorded in controls. Increased Aβ42/40 was also normaliz...

Immunosurgery via IVT-mRNA for the implementation of personalized/precision anticancer-immunotherapy, and tailored circumvention of immunoresistance in non-inflamed or cold-tumors.
Prof Dr Med John Giannios, MD/MBBS, GP, Hon Prof in Medicine, BSc (Biol & Chem), PostCert (Transl Med with Distinction) Med School, Univ of Edinburgh, PostDipl (Transl Med with Distinction) Med School, Univ of Edinburgh, MSc (Transl Med) Med School, Univ of Edinburgh, Post Dipl (Genomic Medicine & Healthcare), Univ of South Wales, Cardiff, Professional Doctorate in Translational CANCER MEDICINE, University of Middlesex, London, UK, Postgraduate Specialty in Family Medicine, Hon President of the Academy of Thermal Medicine, GR, EU, President of the Hel and Int Society of Genomic & Molecular Medicine and Res., GR, EU, ACADEMIC DIRECTOR OF THE POSTGRADUATE PROGRAMME IN GENOMIC (PRECISION) MEDICINE AND HEALTHCARE, UNIVERSITY OF SOUTH WALES, and RECOGNIZED TUTOR IN THE POSTGRADUATE PROGRAMME OF MEDICAL ONCOLOGY OF UNIVERSITY OF BUCKINGHAM, UK.

Immunosurgery utilizing IVT-mRNA for Cancer-Immunotherapy is an innovative therapeutic idea that is merging immune-focused techniques, such as IMMUNOSURGERY with in vitro-transcribed IVT-mRNA for accurately targeting, and eliminating cancer-cells while preserving cells of healthy-tissue.
Immunosurgery consists of a phrase which had been used in developmental-biology for the use of antibodies, and complement for eliminating the outer-layer of an embryo. However, in oncology, it is being referred as the directed destruction of cancer-cells via modified immune-responses consisting of cellular-therapies for immunity, such as CAR-T or TCR-T, antibody-mediated elimination, and targeted-activation of the immune-system which may function as a personalized/precision surgical-instrument.
Very importantly, the in-vitro transcribed-mRNA is an artificially generated-RNA which may be delivered into cells for encoding cancer- markers or molecules which may stimulate the immune-system including cytokines or costimulatory-ligands, and reconfiguring specialized immune-cells, such as T-cells or dendritic-cells.
Its benefits consist of temporary-expression which is secure for DNA, complete inability involving genomic-incorporation, adjustability or  expandability, and mainly utility in mRNA-vaccines, such as the  BioNTech-Pfizer’s COVID19-vaccine.
The merging of IVT-mRNA with immunosurgery is characterized by clinical actionabilities including cancer-vaccines based on mRNA that involves the administration of tumor specific-antigens via mRNA to dendritic-cells or injected directly to cancer-patients for stimulating  cytotoxic T-lymphocytes or CTLs that may lead to the eradication of cancer cells. Analytically, there are tumor-specific (TSA) or tumor associated-antigens (TAAs) which are recognized as neoantigens that refer to proteins resulting from mutations specific to an individual's tumor. Furthermore, synthetic-mRNA may be generated encoding these antigens, and mRNA is containing features that enhance stability, and translation, such as 5’ cap, UTRs or poly(A) tail. The delivery-techniques may include dendritic-cells or DCs which are isolated from the cancer-patient, and they are introduced to mRNA which encodes tumor antigens.
Then these primed-DCs are reinfused into the cancer-patient, and the mRNA may be delivered with lipid-nanoparticles (LNPs) or alternative-carriers via intramuscular or intradermal injection. The antigen-presenting cells may exhibit tumor antigens via MHC class I and II leading to the activation of the immune-system via stimulation of the CD8+ T cells or CTLs, and CD4+ helper T-cells. The In-Situ Immunosurgical-Procedure involves the direct injection of IVT-mRNA into tumors for producing inflammatory-cytokines including IL-12 or IFN-γ, costimulatory-factors, such as CD80 and 86, and antibodies or bispecific T-cell activators (BiTEs). This may generate a pro-inflammatory, and immune-favorable microenvironment resulting in localized-immunosurgery. Furthermore,
IVT-mRNA may also modify immune-cells via temporary-expression of chimeric antigen-receptors (CARs), TCRs specific to tumors, and checkpoint-inhibitors. Subsequently, these immune cells which have been  modified with mRNA may be introduced into the cancer-patient for establishing a precise immune-assault. The subsequent destruction of tumor-cells is achieved by the mRNA regulated immune-pathways which may include program immune-cells, such as T cells or NK-cells. The anticancer-effect of mRNA which is functioning as a precise-tool for immunosurgery may be activated only after the presence of tumor-specific signals.
Generally, IVT mRNA as an artificially created messenger-RNA may be generated externally to the cell by utilizing T7, T3 or SP6 RNA-polymerase, and it is capable of encoding almost any protein including  tumor-antigens, cytokines or costimulatory-molecules. After being delivered into cells via lipid-nanoparticles, dendritic-cell loading or electroporation, the IVT-mRNA may be translated into nearly any type of protein without integrating into the genome. There are companies which are trying to develop mRNA cancer-vaccines against solid-tumors, such as lung, melanoma, etc. Also, research is performed for modifying the tumor-microenvironment or TME so mRNA based IL-12 may be delivered directly into tumor-cells, and IVT mRNA CART-cells are tested against blood-cancers. Delivery-approaches include direct-injection of mRNA- LNPs for vaccines with tumor-antigens, ex-vivo loading of dendritic-cells or DCs with IVT-mRNA for therapeutic-vaccines, and synergistic-combination with checkpoint-inhibitors or adoptive cell-therapy. Their advantages compared to DNA or viral-vectors include absolutely no integration into the host-genome, transient-expression which is limiting systemic-toxicity, rapid and mainly scalable production, and encoding of multiple-payloads in the same formulation composed of cytokines, adjuvants and antigens. 
A huge issue in anticancer treatment consists of immunoresistance which inactivates the pathways of immunotherapy, and it is caused by several mechanisms which are evading the immune-system including tumor-heterogeneity and immunogenic-loss of tumor-antigens because tumor-cells can downregulate the expression of MHC-molecules leading to the prevention of T-cell recognition, T-cell exclusion and/or dysfunction,  immunosuppressive-microenvironment composed of IL-10, TGFβ, TAM, TAN, CAF, NO, ROS, VEGF, FasL, TRAIL, MDSC or Tregs, upregulated immune-checkpoints, such as PDL1, CTLA4-ligands leading to the exhaustion of T-cells, downregulated MHC-I  or major histocompatibility-complex class-I gene that may evade the immune-system in cancer by blocking the presentation of tumor-antigens to CD8+ T-cells.
All these immunoresistance-pathways may be circumvented with the use of IVT-mRNA by antigen-delivery that may cause encoding of multiple-tumor associated-neoantigens for broadening immune-recognition by stimulating CD4+ T-cell, and CD8+ responses which may target multiple tumor-clones or circumvent antigen-loss mutations, genetic-engineering of proteins, such as dominant negative TGFβ-receptors, and  T-cell ex-vivo via mRNA which may upregulate costimulatory-molecules and resist to exhaustion, in-situ reprogramming of the local-immunosuppressive tumor-microenvironment to an inflammatory-state for converting non-inflamed cold to hot immune-inflamed tumors including NSCLC, H&N Ca, CRC, endometrial-Ca, melanoma, bladder and kidney-Ca (RCC)  by delivering via nanoparticles mRNA that encodes specific-chemokines, such as CXCL-9, CXCL-10, CCL-5, IFN-γ, IL-15 or IL-12 for attracting stromal-cells or effector T-cells into cold-tumors which do not elicit strong immune-responses due to poor immune-cell infiltration, and proteins or enzymes which are degrading immunosuppressive-molecules, such as IDO-inhibitors which may be combined with chemo/radio-Tx and immune-checkpoint inhibitors, immune-stimulation by encoding costimulatory-molecules including 4IBBL-ligand or CD137L which is encoded by the TNFSF9-gene or cytokines consisting of  IFNα, GM-CSF or IL12 for boosting dendritic-cells (DCs) or activating T-cells, personalized neoantigen-vaccines mediated by tumor cell-sequencing for identifying patient-specific variants, and encoding as IVT-mRNA vaccines which may lead to the induction of polyclonal T-cell responses  that may circumvent tumor-escape pathways, and checkpoint-modulation for encoding soluble-decoy receptors and/or short lived CRISPR based soluble-immune inhibitors of PD-1, PD-L1 or CTLA-4 in the tumor-microenvironment that may lead directly to transient expression of checkpoint-inhibitors into immune-cells. Furthermore, IVT-mRNA may lead to checkpoint-inhibition via the expression of CARs and/or modified-TCRs which may resist inhibitory-signaling.             
Thus, IVT-mRNA approaches may implement personalized/precision immunotherapy, and circumvent immunoresistance-pathways in cancer via vaccinations, immunomodulation, cell-engineering and gene-editing. Future-challenges include the development of  self amplifying mRNA or saRNAs for prolonging expression, combination of synergistic-treatments, ligands for tumor-targeting on lipid-nanosomes or LNS, prevention of hyperactivation of the innate immune-system by unaltered-mRNA which may require optimized-nucleosides including pseudo-uridine, etc.

Grapevine, as other woody perennials, has been considered a recalcitrant crop to produce transgenic plants. Since the production of transgenic and/or edited plants requires the ability to regenerate plants from transformed tissues, this step is often the biggest bottleneck in the process. The objective of this work is to review the state of the art technologies and strategies for the improvement of grapevine transformation and regeneration, focusing on three aspects: (i) problems associated with grapevine transformation; (ii) genes that promote grapevine regeneration; and (iii) vehicles for gene delivery. Concerning the first aspect, it is well documented that one of the main factors explaining the low success rate in obtaining transgenic plants is the regeneration process. After transgenic integration into receptor cells, tissue culture is required to regenerate transgenic seedlings from transformed cells. This process is time consuming and often requires the addition of environmen...

The molecular technology known as clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is revolutionizing the field of medical research and deepening our understanding of numerous biological processes. The attraction of CRISPR/Cas9 lies in its ability to efficiently edit DNA or modulate gene expression in living eukaryotic cells and organisms, a technology that was once considered either too expensive or scientifically risky. CRISPR/Cas9 has been successfully applied in agriculture to develop the next generation of disease-resistant plants. Now, the capability of gene editing has been translated to the biomedical area, focusing on the future of medicine faced with drug-resistant microbes by selectively targeting genes involved in antibiotic resistance, for example, or finding the ultimate strategy for cancer or HIV. In this regard, it was recently demonstrated that an injection of cancer-fighting CRISPR-modified white blood cells in a patient ...

Overexpression of epithelial cell adhesion molecule (EpCAM) has been implicated in advanced endometrial cancer, but its roles in this progression remain to be elucidated. In addition to its structural role in modulating cell-surface adhesion, here we demonstrate that EpCAM is a regulatory molecule in which its internalization into the nucleus turns on a transcription program. Activation of EGF/EGFR signal transduction triggered cellsurface cleavage of EpCAM, leading to nuclear internalization of its cytoplasmic domain EpICD. ChIP-seq analysis identified target genes that are coregulated by EpICD and its transcription partner, LEF-1. Network enrichment analysis further uncovered a group of 105 genes encoding functions for tight junction, adherent, and cell migration. Furthermore, nanomechanical analysis by atomic force microscopy revealed increased softness and decreased adhesiveness of EGF-stimulated cancer cells, implicating acquisition of an epithelial-mesenchymal transition (EMT) phenotype. Thus, genome editing of EpCAM could be associated with altering these nanomechanical properties towards a less aggressive phenotype. Using this integrative genomic-biophysical approach, we demonstrate for the first time an intricate relationship between EpCAMregulated transcription and altered biophysical properties of cells that promote EMT in advanced endometrial cancer.

We used NOD/SCID mice, also known as NRG, to assess the ability of lentivirus-mediated intravenous delivery of CRISPR in editing the HIV-1 genome from the circulating PBMC engrafts, some of which homed within several animal solid tissues. Lentivirus-mediated delivery of a multiplex of guide RNAs accompanied by Cas9 endonuclease led to the excision of the targeted region of the viral genome positioned within the HIV-1 LTR from the in-vitro-infected human peripheral blood mononuclear cells (PBMCs) embedded in the spleens of NRG mice. Similarly, the treatment of NRG mice harboring PBMC engrafts derived from HIV-1-positive patients with the therapeutic lentivirus eliminated the presence of the viral DNA fragment in the blood, as well as in the spleen, lung, and liver, of the engrafted animals. Sanger sequence analysis of the viral DNA after treatment with the lentiviral vectors expressing Cas9 and gRNAs verified the editing and removal of the proviral DNA fragment from the viral genome ...

CRISPR-associated protein 9 (Cas9)-mediated genome editing provides a promising cure for HIV-1/AIDS; however, gene delivery efficiency in vivo remains an obstacle to overcome. Here, we demonstrate the feasibility and efficiency of excising the HIV-1 provirus in three different animal models using an all-in-one adeno-associated virus (AAV) vector to deliver multiplex single-guide RNAs (sgRNAs) plus Staphylococcus aureus Cas9 (saCas9). The quadruplex sgRNAs/saCas9 vector outperformed the duplex vector in excising the integrated HIV-1 genome in cultured neural stem/progenitor cells from HIV-1 Tg26 transgenic mice. Intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 excised HIV-1 proviral DNA and significantly reduced viral RNA expression in several organs/tissues of Tg26 mice. In EcoHIV acutely infected mice, intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 reduced systemic EcoHIV infection, as determined by live bioluminescence imaging. Additionally, this quadruplex vecto...

The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat off...

Genome editing technologies have revolutionized molecular biology, enabling precise manipulation of gene functions across diverse organisms. In this study, we introduce a novel liposome-mediated delivery system for CRISPR-Cas9 components targeting the Juvenile Hormone Acid Methyltransferase (JHAMT) gene in honey bees (Apis mellifera anatoliaca). This approach leverages drone sperm cells as vectors for CRISPR-Cas9 transfection, overcoming the technical challenges of embryo microinjection in honey bees, such as low survival rates and labor-intensive procedures. The study involved artificial insemination of queen bees with transfected sperm and subsequent evaluation of gene-editing efficiency across generations. Our findings demonstrate the successful generation of both heterozygous and homozygous mutants, with gene-editing efficiencies reaching approximately 43%. This innovative method highlights the potential of liposome-mediated delivery systems for non-invasive, efficient, and scalable genome editing in eusocial insects. The results pave the way for broader applications in honey bee genetic research, offering a viable alternative to traditional methods. Furthermore, this study underscores the importance of genetic tools in advancing apiculture and addressing ecological challenges linked to pollinator health.

Uncertainties in recombinant DNA experimentation remain a significant concern. The potential for off target operation, unintended genetic alterations, unforeseen ecological consequences, and unpredictable effects on human health or inheritance cannot be fully eliminated, despite notable advancements in the field. The long-term ramifications of releasing
genetically modified organisms into the environment, for instance, are not yet thoroughly understood, and the possibility of intergenerational impacts continues to warrant caution.

In order to populate the Moon, Mars, and the Solar System, we will need to take advantage of modern advances in genomics and gene editing to help us survive and thrive. Missions to the International Space Station have seen targeted use of both advanced field gene sequencing devices, and CRISPR-Cas9 for gene editing, to prove the benefits of this technology in spacefaring. To assist in everything from crop growth, radiation resistance, immunology, extreme temperature resistance, and cancer resistance we should take advantage of every facet of genetics. Human genome editing will ultimately be necessary for movement further into the Solar System, but the technology is present to engineer resistance in important organisms whilst the ethics of human gene editing are processing. It is my view that as of the present, not enough discussion is being had in how this can benefit the commercial and governmental sectors, and opportunities are present for further experimentation in research rotation on the ISS and in commercial science missions. Since NASA astronaut Kate Rubins breakthrough missions in genomics and gene editing in 2016 and 2021, there is precedent for further use of CRISPR-Cas9 in microgravity, as well as genomics. Furthermore, recent developments in base editing and prime editing promise more accurate gene editing that should be tested in microgravity to demonstrate the potential benefits of the technology for human survival in the extremes of the Moon, Mars, and the Solar System. In this paper, I summarize the current landscape of genomics and gene editing in space, as well as immediate avenues for advancement in what this technology can do for human Moon, Mars and Solar System missions. I propose that the opportunity is there to solve complex problems for human physiology and food for human population of these extreme environments.

Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a “GG” dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expect...

The blood disorder, β-thalassaemia, is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming peptide nucleic acids (PNAs) substituted at the γ position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human β-thalassaemia. Injection of thalassemic mice with SCF plus nanoparticles containing γPNAs and donor DNAs ameliorated the disease phenotype, with sustained elevation of blood haemoglobin levels into the normal range, reduced reticulocytosis, reversal of splenomegaly and up to 7% β-globin gene correction in HSCs, with extremely low off-target effects. The combination of nanoparticle delivery, next generation γPNAs and SCF treatment may offer a minimally invasive treatment for genetic disorders of the blood that can be achieved safely and sim...

The convergence of cyber and biological threats represents a paradigm shift in how we must approach security. As technology continues to integrate the digital and biological worlds, proactive defense strategies, cross-disciplinary training, and regulatory safeguards will be essential. Investing in secure-by-design biotech and resilient cyber infrastructure is no longer optional—it’s imperative. Learn more in Cyber-Bio Threat Convergence Training by Tonex.

We have observed that of the 10 AAV serotypes, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs), and that the transduction efficiency can be further increased by specifically mutating single surface-exposed tyrosine (Y) residues on AAV6 capsids. In the present studies, we combined the two mutations to generate a tyrosine double-mutant (Y705+731F) AAV6 vector, with which >70% of CD34(+) cells could be transduced. With the long-term objective of developing recombinant AAV vectors for the potential gene therapy of human hemoglobinopathies, we generated the wild-type (WT) and tyrosine-mutant AAV6 vectors containing the following erythroid cell-specific promoters: β-globin promoter (βp) with the upstream hyper-sensitive site 2 (HS2) enhancer from the β-globin locus control region (HS2-βbp), and the human parvovirus B19 promoter at map unit 6 (B19p6). Transgene expression from the B19p6 was significantly higher than that from the HS2-βp, and incre...

Because of CRISPR-Cas9 technology, genome engineering and molecular biology are evolving in new directions. Borrowing from the immune systems of early lifeforms, the CRISPR-Cas9 technology is used to rearrange genetic codes in many organisms precisely. It covers recent developments in CRISPR-Cas9 and highlights its biochemical functions and more uses for this technology. We look into how the structure of the CRISPR-Cas9 complex contributes to guide RNA (gRNA), the use of PAM sequences, and variants of Cas9 such as dCas9, base editors, and prime editors. Recently, RNA editing, epigenetic reprogramming, and diagnostics using CRISPR have been shown to have far more uses than just changing genes. In this area, CRISPR-Cas9 helps to better understand the workings of genes, enhance how metabolic processes function, and change microorganisms for business uses. Using it to help with monogenic conditions, modify the immune system, and deal with infectious diseases has seen fast progress in moving from the lab to patient care. Among the important points in the review are limits like off-target action, difficulty delivering the therapies, and immunogenicity, which continue to block the wide-scale use of gene therapies in medicine. In the last section, the paper outlines what lies ahead, such as using artificial intelligence to guide RNA design, designing CRISPR-based circuits for synthetic biology, and considering the ethics involved with germline editing. Bringing together recent studies, this article points out that CRISPR-Cas9 is becoming increasingly important as a platform for new biotechnology and medicine.

Introduction 2 Delivery of genome editing components into plant cells 3 Delivery methods for genome editing reagents: delivery into single cells 4 Delivery methods for genome editing reagents: delivery into intact tissues 5 Alternatives to DNA delivery 6 Morphogenic genes increase transformation efficiency and extend genotype range 7 Morphogenic genes permit transformation in nontraditional explants 8 Future trends 9 Where to look for further information 10 References

Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide a strategy for characterization of dose-dependent effector functions of essential gen...

Metastatic renal cell carcinoma (mRCC) is nearly incurable and accounts for most of the mortality associated with RCC. Von Hippel Lindau (VHL) is a tumour suppressor that is lost in the majority of clear cell RCC (ccRCC) cases. Its role in regulating hypoxia-inducible factors-1α (HIF-1α) and -2α (HIF-2α) is well-studied. Recent work has demonstrated that VHL knock down induces an epithelial-mesenchymal transition (EMT) phenotype. In this study we showed that a CRISPR/Cas9-mediated knock out of VHL in the RENCA model leads to morphologic and molecular changes indicative of EMT, which in turn drives increased metastasis to the lungs. RENCA cells deficient in HIF-1α failed to undergo EMT changes upon VHL knockout. RNA-seq revealed several HIF-1α-regulated genes that are upregulated in our VHL knockout cells and whose overexpression signifies an aggressive form of ccRCC in the cancer genome atlas (TCGA) database. Independent validation in a new clinical dataset confirms the upregulation...

Editing human germline genes may act as a boon in certain genetics and other illnesses. Specifically, through groundbreaking technologies like CRISPR-Cas9, gene editing has rapidly evolved from a scientific possibility to a practical tool with immense potential. It offers transformative benefits by allowing precise alterations to DNA, particularly in fields like medicine and agriculture. However, the technology raises profound ethical questions that touch on the very fabric of human existence, social equity, and the natural world. This article explores the ethical considerations surrounding gene editing, offering a balanced perspective on its promise and potential pitfalls.

Plants or plant cells can be used to produce pharmacological glycoproteins such as antibodies or vaccines. However these proteins carry N-glycans with plant-typical residues [β(1,2)-xylose and core α(1,3)-fucose], which can greatly impact the immunogenicity, allergenicity, or activity of the protein. Two enzymes are responsible for the addition of plant-specific glycans: β(1,2)-xylosyltransferase (XylT) and α(1,3)-fucosyltransferase (FucT). Our aim consisted of knocking-out two XylT genes and four FucT genes (12 alleles altogether) in Nicotiana tabacum BY-2 suspension cells using CRISPR/Cas9. Three XylT and six FucT sgRNAs were designed to target conserved regions. After transformation of N. tabacum BY-2 cells with genes coding for sgRNAs, Cas9, and a selectable marker (bar), transgenic lines were obtained and their extracellular as well as intracellular protein complements were analyzed by Western blotting using antibodies recognizing β(1,2)-xylose and α(1,3)-fucose. Three lines sh...

Durum-21 (D-21) is a high-yielding, disease resistant with better-quality traits variety, developed by Wheat Research Institute Faisalabad. This variety is mainly developed for industrial purposes for pasta production. Worldwide, durum wheat is utilized for pasta production; but in Pakistan, due to a lack of research work and non-availability of quality seeds of durum wheat, bread wheat is being utilized for pasta production. D-21 is developed with the breeding code of D-21 having parentage of FKN/3/2*FR//KAD/GB/4/BB/CHA/5/AS-2002 with pedigree as PB20733-1a-2a-2a-0a-0a-19a-0a. The candidate line D-21 was developed by crossing a germplasm accession with approved bread wheat variety AS-02. The genotype was further evaluated over multiple locations in Punjab Pakistan for yield and yield-related attributes in the station, provincial, and national uniform durum yield trials executed by Wheat Research Institute (WRI), Faisalabad during 2015-20. The promising line out yielded the two comm...

341 entries comprising of 250 genotypes/lines and 91 gene differentials were tested for leaf rust (Puccinia triticina Erik) in different ecological zones of Punjab during 2016–17 and 2017–18. Each entry was planted in a single 1 m long row and Morocco was used as a spreader. Data on leaf rust severity was recorded once in 3rd week of March during both study years at all locations by following Modified Cobb Scale while the data was recorded three times on 2nd, 22nd and 29th March during 2018 at Faisalabad location to study rust development pattern. The disease severity ranged from 0-100S during 2016–17 and from 0-80S during 2017–18. The genotype HYT 60–5 and the genes Lr-19, Lr-26 and Lr 27+31 showed no disease symptoms at any location during both the study years. These genes can be used for future breeding material development. Area under disease progressive curve (AUDPC), calculated on the basis of periodical readings from Faisalabad, ranged from 0–550 and the susceptible check Mor...

This article explores the new developments and challenges of agricultural Gene Editing (GED) regulation in primarily nine countries of Latin America and the Caribbean (LAC) Region: Argentina, Bolivia, Brazil, Colombia, Guatemala, Honduras, Mexico, Paraguay and Peru. As Gene Editing technology develops, Latin America and the Caribbean regulatory regimes struggle to keep pace. Developers and regulators face challenges such as consumer perceptions, intellectual property, R&D funding (private and public), training, environmental and social impact, and access to domestic and international markets. Some Latin America and the Caribbean countries (e.g., Argentina) interpret existing legislation to promulgate regulations for biotechnology and Genetically Modified Organisms (GMOs), while others (e.g., Brazil and Honduras) have specific legislation for Genetically Modified Organisms. In both those cases, often a case-by-case approach is chosen to determine whether a Gene Editing organism is su...

The spatiotemporal control of 3D genome is fundamental for gene regulation, yet it remains challenging to profile high-resolution chromatin structure at cis-regulatory elements (CREs). Using C-terminally biotinylated dCas9, endogenous biotin ligases, and pooled sgRNAs, we describe the dCas9-based CAPTURE method for multiplexed analysis of locus-specific chromatin interactions. The redesigned system allows for quantitative analysis of the spatial configuration of a few to hundreds of enhancers or promoters in a single experiment, enabling comparisons across CREs within and between gene clusters. Multiplexed analyses of the spatiotemporal configuration of erythroid super-enhancers and promoter-centric interactions reveal organizational principles of genome structure and function.

In individuals with Duchenne muscular dystrophy (DMD), exon skipping treatment to restore a wild-type phenotype or correct the frame shift of the mRNA transcript of the dystrophin (DMD) gene are mutation-specific. To explore the molecular characterization of DMD rearrangements and predict the reading frame, we simultaneously screened all 79 DMD gene exons of 45 unrelated male DMD patients using a multiplex ligation-dependent probe amplification (MLPA) assay for deletion/duplication patterns. Multiplex PCR was used to confirm single deletions detected by the MLPA. There was an obvious diagnostic delay, with an extremely statistically significant difference between the age at initial symptoms and the age of clinical evaluation of DMD cases (t value, 10.3; 95% confidence interval 5.95-8.80, P < 0.0001); the mean difference between the two groups was 7.4 years. Overall, we identified 147 intragenic rearrangements: 46.3% deletions and 53.7% duplications. Most of the deletions (92.5%) ...

It is well established that antiretroviral therapy (ART), while highly effective in controlling HIV replication, cannot eliminate virus from the body. Therefore, the majority of HIV-1-infected individuals remain at risk for developing AIDS due to persistence of infected reservoir cells serving as a source of virus re-emergence. Several reservoirs containing replication competent HIV-1 have been identified, most notably CD4+ T cells. Cells of the myeloid lineage, which are the first line of defense against pathogens and participate in HIV dissemination into sanctuary organs, also serve as cellular reservoirs of HIV-1. In latently infected resting CD4+ T cells, the integrated copies of proviral DNA remain in a dormant state, yet possess the ability to produce replication competent virus after cellular activation. Studies have demonstrated that modification of chromatin structure plays a role in establishing persistence, in part suggesting that latency is, controlled epigenetically. Current efforts to eradicate HIV-1 from this cell population focus primarily on a "shock and kill" approach through cellular reactivation to trigger elimination of virus producing cells by cytolysis or host immune responses. However, studies revealed several limitations to this approach that require more investigation to assess its clinical application. Recent advances in gene editing technology prompted use of this approach for inactivating integrated proviral DNA in the genome of latently infected cells. This technology, which requires a detailed understanding of the viral genetics and robust delivery, may serve as a powerful strategy to eliminate the latent reservoir in the host leading to a sterile cure of AIDS.

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in FBN1 gene, which encodes a key extracellular matrix protein FIBRILLIN-1. The haplosufficiency of FBN1 has been implicated in pathogenesis of MFS with manifestations primarily in cardiovascular, muscular, and ocular tissues. Due to limitations in animal models to study the late-onset diseases, human pluripotent stem cells (PSCs) offer a homogeneic tool for dissection of cellular and molecular pathogenic mechanism for MFS in vitro. Here, we first derived induced PSCs (iPSCs) from a MFS patient with a FBN1 mutation and corrected the mutation, thereby generating an isogenic "gain-of-function" control cells for the parental MFS iPSCs. Reversely, we knocked out FBN1 in both alleles in a wild-type (WT) human embryonic stem cell (ESC) line, which served as a loss-of-function model for MFS with the WT cells as an isogenic control. Mesenchymal stem cells derived from both FBN1-mutant iPSCs and -ESCs demonst...

Genome editing using the CRISPR/Cas9 RNA-guided endonuclease system has rapidly become a driving force for discovery in modern biomedical research. This simple yet elegant system has been widely used to generate both loss-of-function alleles and precision knock-in mutations using single-stranded donor oligonucleotides. Our CRISPRtools platform supports both of these applications in order to facilitate the use of CRISPR/Cas9. While there are several tools that facilitate CRISPR/Cas9 design and screen for potential off-target sites, the process is typically performed sequentially on single genes, limiting scalability for large-scale programs. Here, the design principle underlying gene ablation is based upon using paired guides flanking a critical region/exon of interest to create deletions. Guide pairs are rank ordered based upon published efficiency scores and off-target analyses, and reported in a concise format for downstream implementation. The exon deletion strategy simplifies ch...

The state-of-the-art laboratory at hand indicates that, concerning Polycystic Kidney Disease (PKD), nothing is impossible to manage using gene-editing tools, such as CRISPR-Cas9. PKD is a disorder in which the fluid-filled sacs develop within the kidneys, enlarging them and impairing their activity. New advances in CRISPR-Cas9 editing, which allows targeting particular parts of the DNA, provide access to eliminating the mutations that lead to the development of PKD. The trick is that it is not yet locked in as far as success is concerned-it is stated that in certain studies, better results have been achieved working on the PKD1 and PKD2 genes, as compared to the others. Such incoherence portrays the emergence of new technical challenges. Experts' conversations stress the need to come to an agreement on how CRISPR/Cas9 should be used on human embryos. This paper discusses the development of CRISPR/Cas9 as well as its application to control polycystic kidney disease (PKD), and the ethical implications thereof. Going through the previous work and contemporary discussion, the paper will explain what CRISPR/Cas9 technology would offer to PKD and highlight the limitations of the technology, contributing to the overall discussion of whether gene editing has a potentially bright future in healthcare.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene variant, c.3453G > C (D1152H), is associated with mild Cystic Fibrosis (CF) disease, though there is considerable clinical variability ranging from no detectable symptoms to lung disease with early acquisition of Pseudomonas aeruginosa. The approval extension of ivacaftor, the first CFTR modulator drug approved, to include D1152H was based on a positive drug response of defective CFTR-D1152H chloride channel function when expressed in FRT cells. Functional analyses of primary human nasal epithelial cells (HNE) from an individual homozygous for D1152H now revealed that while CFTR-D1152H demonstrated normal, wild-type level chloride conductance, its bicarbonate-selective conductance was impaired. Treatment with ivacaftor increased this bicarbonate-selective conductance. Extensive genetic, protein and functional analysis of the nasal cells of this D1152H/D1152H patient revealed a 90% reduction of CFTR transcripts du...

The combination therapy of lumacaftor and ivacaftor (Orkambi(®)) is approved for patients bearing the major cystic fibrosis (CF) mutation: ΔF508 It has been predicted that Orkambi(®) could treat patients with rarer mutations of similar "theratype"; however, a standardized approach confirming efficacy in these cohorts has not been reported. Here, we demonstrate that patients bearing the rare mutation: c.3700 A>G, causing protein misprocessing and altered channel function-similar to ΔF508-CFTR, are unlikely to yield a robust Orkambi(®) response. While in silico and biochemical studies confirmed that this mutation could be corrected and potentiated by lumacaftor and ivacaftor, respectively, this combination led to a minor in vitro response in patient-derived tissue. A CRISPR/Cas9-edited bronchial epithelial cell line bearing this mutation enabled studies showing that an "amplifier" compound, effective in increasing the levels of immature CFTR protein, augmented t...

Argentina currently has a regulation for genome-editing products whose criteria were updated as consultations were received to determine the regulatory status of these products. The aim of this regulation is to consider all organisms (animals, micro-organisms and plants) under the same NBT resolution independently and without being linked to commercial Genetically Modified Organism (GMO) regulations. This gives certainty to local researchers and developers (teams of local developers and researchers), which can be seen in the number of developments and consultations carried out. It should be noted that early results showed that the speed of innovation of these technologies was increasing in a short time, giving more opportunity to local developers who showed interest in generating products in different species, crops and phenotypes.

The rapid development of genome modification technology has provided many great benefits in diverse areas of research and industry. Genome modification technologies have also been actively used in a variety of research areas and fields of industry in avian species. Transgenic technologies such as lentiviral systems and piggyBac transposition have been used to produce transgenic birds for diverse purposes. In recent years, newly developed programmable genome editing tools such as transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) have also been successfully adopted in avian systems with primordial germ cell (PGC)-mediated genome modification. These genome modification technologies are expected to be applied to practical uses beyond system development itself. The technologies could be used to enhance economic traits in poultry such as acquiring a disease resistance or p...

The new reproductive technologies have opened the door to different processes of germline genetic enhancement by which the characteristics of an individual according to the interests of the agents involved could be selected during its gestation. Although the initiative is apparently oriented towards developing individuals that would excel in society, critical voices raise the concerns about that this approach would generate and need for a reflection on the ethical, social and legal implications of these techniques and their implementation in society. We reviewed the literature about these issues throughout their historical records to date, focusing on the moral arguments and non-clinical aspects that affect the legal and social environment. We have observed various trends of thought with divergent positions (proactive, preventive, and regulatory) as well as a large number of articles that try to reconcile the different approaches. This review illustrates a series of concepts from the ethics and philosophy fields which are frequently used in studies that evaluate the ethical implications of germline genetic enhancement, such as dignity, benefit, autonomy, and identity. In addition, amongst the many unresolved controversies surrounding genetic enhancement, we identify procreative beneficence, genetic disassociation, gender selection, the value of disability, embryo chimerization, and the psychosocial inequality of potentially enhanced individuals as crucial. We also develop possible scenarios for future debate. We consider especially important the definition and specification of three aspects which are essential for the deployment of new reproductive technologies: the moral status of the embryo undergoing enhancement, the legal status of the enhanced individual, and the responsibility of the agents executing the enhancement. Finally, we propose the precautionary principle as a means to navigate ethical uncertainties.

DNA holds genetic information in the nucleus of eukaryotic cells; and has three different functions: replication, storage of hereditary information, and regulation of cell division. Most studies described the association of single nucleotide polymorphism (SNP) to common orthopaedics diseases and the susceptibility to develop musculoskeletal injuries. Several mutations are associated with osteoporosis, musculoskeletal ailments and other musculoskeletal deformity and conditions. Several strategies, including gene therapy and tissue engineering with mesenchymal stem cells (MSC), have been proposed to enhance healing of musculoskeletal tissues. Furthermore, a recent technique has revolutionized gene editing: clustered regulatory interspaced short palindromic repeat (CRISPR) technology is characterized by simplicity in target design, affordability, versatility, and high efficiency, but needs more studies to become the preferred platform for genome editing. Predictive genomics DNA profili...

Despite the success of antiretroviral therapy (ART), HIV remains incurable due to the persistence of latent viral reservoirs that evade immune clearance and therapy. Current strategies targeting latency, such as "shock-and-kill" and "block-and-lock," have shown limited success and safety concerns. We propose a novel phased gene therapy approach using engineered HIV-derived lentiviral vectors to target both actively replicating and latent HIV. In the first phase, a Therapeutic Interfering Particle (TIP) or antiviral gene cassette is delivered to suppress active HIV replication. In the second phase, following suppression, a latency-reversing or cytotoxic payload is introduced via a second vector to reactivate and eliminate reservoir cells. This staged model leverages the natural tropism and replication biology of HIV to deliver therapy precisely and sequentially. If validated, this strategy could offer a path toward a functional or sterilizing cure for HIV infection.

This paper aims to do an interdisciplinary exercise between gene editing and theology, especially considering the encyclical Laudato Si’. It seeks an expanded reason in a two-way direction: genome editing open to theology, and theology open to genetic knowledge. First, this research analyses how gene editing can broaden its horizons and allow itself to be challenged by Laudato Si’s reflections. A parallel is drawn between the ten axes of Laudato Si’ and the global governance of gene editing. Secondly, this work discusses how theology, as observed in Laudato Si’, can further expand its horizons and be enriched by the possibilities of gene editing. Specifically, the six numbers of this encyclical letter that speak directly about genetics are considered. Subsequently, some issues related to gene editing not covered in Laudato Si’ are presented, such as the de-extinction of some species, the use of gene drives in disease-transmitting species, gene editing in animals for transplantation, advances in hybrids and chimaeras, the rise of biohackers. The results of open reason on the part of both gene editing and theology bring new and crucial questions that arise from the encounter of these two fields of knowledge.

Molecular genetics explores the molecular mechanisms of genetic information and its expression. It integrates concepts from genetics, biochemistry, and molecular biology to understand the structure, function, and regulation of genes at the molecular level. Key topics include DNA structure and replication, RNA transcription and processing, protein synthesis, gene regulation, and the study of mutations and their effects. Molecular genetics has revolutionized our understanding of heredity, disease, and evolution, providing tools for genetic engineering, diagnostics, and therapeutic interventions.

Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can ...

Over the last decade, technological advances in genomics have generated an unprecedented wealth of new information on underlying defects, mechanisms, and therapeutic targets of skin diseases. They have led to the development of biological systems to profile and understand gene, RNA, and protein networks and biological cascades in disease models and in patients. Functional genomics has shown complex webs of interactions, and integrative biology has become essential to capturing, comparing, and understanding these networks. Transcriptomics allows deciphering of disease signatures and signaling networks to discover mechanisms for disease variability and to define groups of skin conditions with shared functional profiles, which may help redefine a new nosology of skin diseases . Technological advances in mass spectrometry-based proteomics now allow measurement of changes in protein abundance, posttranslational modifications, and proteinprotein interactions at the scale of the proteome, resulting in the identification of mechanisms of skin diseases and targets for therapeutic intervention . Epigenomics, epistasis, metabolome, microbiome, and phenomics are also being developed to account for the complexity of biosystems and to set up the bases for integrative genomics . Genomic medicine could lead to a more comprehensive understanding of skin diseases, to personalized medicine, and to improved knowledge of genotype-phenotype interactions and is essential for making new medicines. In fact, spectacular progress has been made in targeted therapies derived from improved understanding of the disease pathogenesis. Some of the best examples have led to the development of specific and highly effective small-molecule drugs blocking the disease biological cascade, and others have used gene therapy or cell therapy to replace the function of a defective gene, to provide cells with high potential to reverse the phenotype, or to repurpose existing drugs. Next-generation sequencing (Metzker, 2010) has been instrumental in identifying new disease-causing genes and unfolding the genetic landscape of a number of genetic skin diseases. Next-generation sequencing brings a new paradigm for the geneticist, allowing for the identification of Mendelian genes from very rare diseases and/or when no large families are available. However, the rapid identification in an