Cas9 Gene Editing Research Articles

IFIT3 mediates TBK1 phosphorylation to promote activation of pDCs and exacerbate systemic sclerosis in mice.

To assess the impact of the IFIT3/TBK1 signalling pathway in activating plasmacytoid dendritic cells (pDCs) and its role in the development of SSc. Utilized single-cell RNA sequencing (scRNA-seq) and high-throughput transcriptome RNA sequencing to reveal the differential abundance of pDCs and the role of the key gene IFIT3 in SSc. Conducted in vitro cell experiments to evaluate the effect of IFIT3/TBK1 signalling pathway intervention on pDC activation cytokine release and fibroblast function. Constructed an IFIT3-/- mouse model using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing to assess the potential benefits of intervening in the IFIT3/TBK1 signalling pathway on skin and lung fibrosis in the SSc mouse model. The IFIT3/TBK1 signalling pathway plays a crucial role in activating pDCs, with IFIT3 acting as an upstream regulator of TBK1. Intervention in the IFIT3/TBK1 signalling pathway can inhibit pDC activation cytokine release and impact fibroblast function. The IFIT3-/- mouse model shows potential benefits of targeting the IFIT3/TBK1 signalling pathway in reducing skin and lung fibrosis in the SSc mouse model. This study provides new insights into potential therapeutic targets for SSc, highlighting the critical role of the IFIT3/TBK1 signalling pathway in SSc development. This study elucidates the pivotal role of plasmacytoid dendritic cells (pDCs) in systemic sclerosis (SSc). This study identified the key regulatory gene involved in systemic sclerosis (SSc) as IFIT3. This study has found that IFIT3 functions as an upstream regulatory factor, activating TBK1. This study provides Evidence of the regulatory effects of the IFIT3/TBK1 pathway on plasmacytoid dendritic cells (pDCs). This study validated the therapeutic potential using the IFIT3-/- mouse model.

Strategies of Bladder Reconstruction after Partial or Radical Cystectomy for Bladder Cancer.

The standard strategy is to reconstruct bladder by use of bowel segments as material in bladder cancer with radical cystectomy clinically. Both natural derived and non natural derived materials are investigated in bladder reconstruction. Studies on mechanical bladder, bladder transplantation and bladder xenotransplantation are currently limited although heart and kidney transplantation or xenotransplantation are successful to a certain extent, and bone prostheses are applied in clinical contexts. Earlierlimited number of studies associated with bladder xenograft from animals to humans were notparticular promisingin results. Although there have beeninvestigations on pig to human cardiac xenotransplantation with CRISPR Cas9 gene editing, theCRISPR Cas techniqueisnot yet widelyresearchedin porcinebladder related gene editingfor the potential of human bladder replacementfor bladder cancer. The advancement of technologies such as gene editing, bioprinting and induced pluripotent stem cells allow further research into partial or wholebladder replacement strategies. Porcine bladder is suggested as a potential source material for bladder reconstruction due to its alikeness to human bladder. Challenges thatexist with all these approaches need to be overcome. This paper aims to review gene editing technology such as the CRISPR Cas systems as tools in bladder reconstruction, bladder xenotransplantation and hybrid bladder with technologies of induced pluripotent stem cells and genome editing, bioprinting for bladder replacement for bladder reconstruction and to restore normal bladder control function after cystectomy for bladder cancer.

Secondary Conformational Checkpoint in CRISPR-Cas9.

A specific checkpoint between target DNA binding and cleavage primarily governs the precision of Cas9 gene editing. Although various CRISPR-Cas9 variants have been developed to improve DNA cleavage accuracy, we still lack a comprehensive understanding of how they work at the molecular level. Herein, we have focused on studying the late-stage conformational transitions of Cas9 and an evolved Cas9 mutant (evoCas9) that start from the precleavage state. Our submilliseconds of dynamic simulations reveal that the presence of base mismatches leads the HNH nuclease domain of Cas9 to alter its principal functional modes of motion, thereby impairing its conformational activation. This observation suggests the existence of a secondary conformational checkpoint that fine-tunes the final DNA cleavage activation. Remarkably, evoCas9 is prone to deviating from the normal activation pathway with base mismatches. This is characterized by a noticeable shift in the positioning of the HNH domain and a significantly perturbed allosteric communication network within the enzyme. Therefore, the mutations evolved in evoCas9 also reinforce the secondary checkpoint in addition to the previously identified primary checkpoint, collectively ensuring this variant's high gene-editing accuracy. This mechanism should also apply to other Cas9-guide RNA variants with enhanced fidelity.

Exagamglogene Autotemcel for Transfusion-Dependent β-Thalassemia

BackgroundExagamglogene autotemcel (exa-cel) is a nonviral cell therapy designed to reactivate fetal hemoglobin synthesis through ex vivo clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 gene editing of the erythroid-specific enhancer region of BCL11A in autologous CD34+ hematopoietic stem and progenitor cells (HSPCs).MethodsWe conducted an open-label, single-group, phase 3 study of exa-cel in patients 12 to 35 years of age with transfusion-dependent β-thalassemia and a β0/β0, β0/β0-like, or non–β0/β0-like genotype. CD34+ HSPCs were edited by means of CRISPR-Cas9 with a guide mRNA. Before the exa-cel infusion, patients underwent myeloablative conditioning with pharmacokinetically dose-adjusted busulfan. The primary end point was transfusion independence, defined as a weighted average hemoglobin level of 9 g per deciliter or higher without red-cell transfusion for at least 12 consecutive months. Total and fetal hemoglobin concentrations and safety were also assessed.ResultsA total of 52 patients with transfusion-dependent β-thalassemia received exa-cel and were included in this prespecified interim analysis; the median follow-up was 20.4 months (range, 2.1 to 48.1). Neutrophils and platelets engrafted in each patient. Among the 35 patients with sufficient follow-up data for evaluation, transfusion independence occurred in 32 (91%; 95% confidence interval, 77 to 98; P<0.001 against the null hypothesis of a 50% response). During transfusion independence, the mean total hemoglobin level was 13.1 g per deciliter and the mean fetal hemoglobin level was 11.9 g per deciliter, and fetal hemoglobin had a pancellular distribution (≥94% of red cells). The safety profile of exa-cel was generally consistent with that of myeloablative busulfan conditioning and autologous HSPC transplantation. No deaths or cancers occurred.ConclusionsTreatment with exa-cel, preceded by myeloablation, resulted in transfusion independence in 91% of patients with transfusion-dependent β-thalassemia. (Supported by Vertex Pharmaceuticals and CRISPR Therapeutics; CLIMB THAL-111 ClinicalTrials.gov number, NCT03655678.)

Exagamglogene Autotemcel for Severe Sickle Cell Disease

BackgroundExagamglogene autotemcel (exa-cel) is a nonviral cell therapy designed to reactivate fetal hemoglobin synthesis by means of ex vivo clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 gene editing of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) at the erythroid-specific enhancer region of BCL11A.MethodsWe conducted a phase 3, single-group, open-label study of exa-cel in patients 12 to 35 years of age with sickle cell disease who had had at least two severe vaso-occlusive crises in each of the 2 years before screening. CD34+ HSPCs were edited with the use of CRISPR-Cas9. Before the exa-cel infusion, patients underwent myeloablative conditioning with pharmacokinetically dose-adjusted busulfan. The primary end point was freedom from severe vaso-occlusive crises for at least 12 consecutive months. A key secondary end point was freedom from inpatient hospitalization for severe vaso-occlusive crises for at least 12 consecutive months. The safety of exa-cel was also assessed.ResultsA total of 44 patients received exa-cel, and the median follow-up was 19.3 months (range, 0.8 to 48.1). Neutrophils and platelets engrafted in each patient. Of the 30 patients who had sufficient follow-up to be evaluated, 29 (97%; 95% confidence interval [CI], 83 to 100) were free from vaso-occlusive crises for at least 12 consecutive months, and all 30 (100%; 95% CI, 88 to 100) were free from hospitalizations for vaso-occlusive crises for at least 12 consecutive months (P<0.001 for both comparisons against the null hypothesis of a 50% response). The safety profile of exa-cel was generally consistent with that of myeloablative busulfan conditioning and autologous HSPC transplantation. No cancers occurred.ConclusionsTreatment with exa-cel eliminated vaso-occlusive crises in 97% of patients with sickle cell disease for a period of 12 months or more. (CLIMB SCD-121; ClinicalTrials.gov number, NCT03745287.)

Polymeric micellar nanoparticles for effective CRISPR/Cas9 genome editing in cancer

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) gene editing has attracted extensive attentions in various fields, however, its clinical application is hindered by the lack of effective and safe delivery system. Herein, we reported a cationic micelle nanoparticle composed of cholesterol-modified branched small molecular PEI (PEI-CHO) and biodegradable PEG-b-polycarbonate block copolymer (PEG-PC), denoted as PEG-PC/PEI-CHO/pCas9, for the CRISPR/Cas9 delivery to realize genomic editing in cancer. Specifically, PEI-CHO condensed pCas9 into nanocomplexes, which were further encapsulated into PEG-PC nanoparticles (PEG-PC/PEI-CHO/pCas9). PEG-PC/PEI-CHO/pCas9 had a PEG shell, protecting DNA from degradation by nucleases. Enhanced cellular uptake of PEG-PC/PEI-CHO/pCas9 nanoparticles was observed as compared to that mediated by Lipo2k/pCas9 nanoparticles, thus leading to significantly elevated transfection efficiency after escaping from endosomes via the proton sponge effect of PEI. In addition, the presence of PEG shell greatly improved biocompatibility, and significantly enhanced the in vivo tumor retention of pCas9 compared to PEI-CHO/pCas9. Notably, apparent downregulation of GFP expression could be achieved both in vitro and in vivo by using PEG-PC/PEI-CHO/pCas9-sgGFP nanoparticles. Furthermore, PEG-PC/PEI-CHO/pCas9-sgMcl1 induced effective apoptosis and tumor suppression in a HeLa tumor xenograft mouse model by downregulating Mcl1 expression. This work may provide an alternative paradigm for the efficient and safe genome editing in cancer.

Traditional and emerging strategies using hepatocytes for pancreatic regenerative medicine.

Although pancreas and islet cell transplantation are the only ways to prevent the late complications of insulin-dependent diabetes, a shortage of donors is a major obstacle to tissue and organ transplantation. Stem cell therapy is an effective treatment for diabetes and other pancreatic-related diseases, which can be achieved by inducing their differentiation into insulin-secreting cells. The liver is considered an ideal source of pancreatic cells due to its similar developmental origin and strong regenerative ability as the pancreas. This article reviews the traditional and emerging strategies using hepatocytes for pancreatic regenerative medicine and evaluates their advantages and challenges. Gene reprogramming and chemical reprogramming technologies are traditional strategies with potential to improve the efficiency and specificity of cell reprogramming and promote the transformation of hepatocytes into islet cells. At the same time, organoid technology, as an emerging strategy, has received extensive attention. Biomaterials provide a three-dimensional culture microenvironment for cells, which helps improve cell survival and differentiation efficiency. In addition, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology has brought new opportunities and challenges to the development of organoid technology.

Application and progress of CRISPR/Cas9 gene editing in B-cell lymphoma: a narrative review.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) gene editing and CRISPR/Cas9 screening libraries are hot topics, and have high application values in the diagnosis and treatment of genetic diseases, and the improvement of prognosis. The major treatment of B-cell lymphoma is chemotherapy combined with biological therapy. Due to the individual specificity and the emergence of drug resistance, the therapeutic efficacy varies. The objective of this article is to explore potential targets to enhance therapeutic effects, optimize treatment plans, and improve the prognosis of patients with B-cell lymphoma. We undertook a comprehensive, narrative review of the latest literature to define the current application and progress of CRISPR/Cas9 in B-cell lymphoma. The concepts of CRISPR/Cas9, the mechanism of gene editing, and the procedures of CRISPR/Cas9 screening libraries are investigated for candidate genes. We mainly focus on application and progress of CRISPR/Cas9 in B-cell lymphoma and screen out some genes, signaling pathways, and cytokines, which may become potential targets for clinical treatment. CRISPR/Cas9 gene editing has great promise in the treatment of B-cell lymphoma. This article reviews some genes, signaling pathways, and cytokines related to the progression and prognosis of B-cell lymphoma to provide a strong theoretical basis.

The Potential Therapeutic Applications of CRISPR/Cas9 in the Treatment of Gastrointestinal Cancers.

Gastrointestinal (GI) cancer is one the most prevalent types of cancer. Despite current chemotherapy's success, patients with GI cancer continue to have a dismal outcome. The onset and progression of cancer are caused by alterations and the abnormal expression of several families of genes, like tumor-suppressor genes, oncogenes, and chemotherapy-resistant genes. The final purpose of tumor therapy is to inhibit cellular development by modifying mutations and editing the irregular expression of genes It has been reported that CDH1, TP53, KRAS, ARID1A, PTEN, and HLA-B are the commonly mutated genes in GI cancer. Gene editing has become one potential approach for cases with advanced or recurrent CRC, who are nonresponsive to conventional treatments and a variety of driver mutations along with progression cause GI progression. CRISPR/Cas9 technique is a reliable tool to edit the genome and understand the functions of mutations driving GI cancer development. CRISPR/Cas9 can be applied to genome therapy for GI cancers, particularly with reference to molecular-targeted medicines and suppressors. Moreover, it can be used as a therapeutic approach by knocking in/out multiple genes. The use of CRISPR/ Cas9 gene editing method for GI cancer therapy has therefore resulted in some improvements. There are several research works on the role of CRISPR/Cas9 in cancer treatment that are summarized in the following separate sections. Here, the use of CRISPR/Cas9-based genome editing in GI and the use of CRISPR/Cas9 is discussed in terms of Targeting Chemotherapy Resistance-related Genes like; KRAS, TP53, PTEN, and ARID1A.

Genomic blueprints of soybean (Glycine max) pathogen resistance: revealing the key genes for sustainable agriculture.

Soybean (Glycine max ) is an important oilseed, protein and biodiesel crop. It faces significant threats from bacterial, fungal and viral pathogens, which cause economic losses and jeopardises global food security. In this article, we explore the relationship between soybeans and these pathogens, focusing on the molecular responses that are crucial for soybeans defence mechanisms. Molecular responses involve small RNAs and specific genes, including resistance (R) genes that are pivotal in triggering immune responses. Functional genomics, which makes use of cutting-edge technologies, such as CRISPR Cas9 gene editing, allows us to identify genes that provide insights into the defence mechanisms of soybeans with the focus on using genomics to understand the mechanisms involved in host pathogen interactions and ultimately improve the resilience of soybeans. Genes like GmKR3 and GmVQ58 have demonstrated resistance against soybean mosaic virus and common cutworm, respectively. Genetic studies have identified quantitative trait loci (QTLs) including those linked with soybean cyst nematode, root-knot nematode and Phytophthora root and stem rot resistance. Additionally, resistance against Asian soybean rust and soybean cyst nematode involves specific genes and their variations in terms of different copy numbers. To address the challenges posed by evolving pathogens and meet the demands of a growing population, accelerated soybean breeding efforts leveraging functional genomics are imperative. Targeted breeding strategies based on a deeper understanding of soybean gene function and regulation will enhance disease resistance, ensuring sustainable agriculture and global food security. Collaborative research and continued technological advancements are crucial for securing a resilient and productive agricultural future.

Production of a PSEN1 genetically engineered marmoset model of early‐onset Alzheimer’s Disease

AbstractBackgroundThe lack of an adequate animal model of Alzheimer’s disease (AD) that mirrors the natural onset and progression of the human disorder has been a substantial barrier to basic and translational research into AD. Consequently, many fundamental questions remain about the key mechanisms that initiate the disease and the factors that promote its progression. While rodents are valuable AD models, nonhuman primates (NHPs) offer many advantages and have greater translational relevance. The common marmoset (Callithrix jacchus) is a small, prolific New World NHP that is amenable to genetic engineering and is rapidly becoming a valuable model organism, especially for neuroscience research.MethodWe used CRISPR/Cas9 technology to generate marmosets with C410Y or A426P point mutations in the PSEN1 gene. In humans, these mutations lead to familial early‐onset autosomal dominant Alzheimer’s Disease. Briefly, we designed, synthesized, and injected CRISPR/Cas9 reagents into single‐cell embryos that were subsequently transferred to synchronized recipient females. One adult C410Y founder animal was bred to a wild‐type mate.ResultTo date, we have produced seven live‐born marmosets harboring the PSEN1 C410Y (3 males, 1 female) or A426P (1 male, 2 females) mutations. One male founder animal, a compound heterozygote harboring C410Y and 1bp insertion PSEN1 alleles, has successfully sired three litters and transmitted the C410Y substitution (n = 6) and the 1bp insertion (n = 5) alleles to F1 offspring. We are currently characterizing the founders and F1 offspring using fluid‐based biomarkers, non‐invasive neuroimaging, and behavioral assessment to identify early markers of disease progression.ConclusionCRISPR/Cas9 gene editing was used to successfully establish a line of genetically engineered marmosets expected to model early‐onset AD. This nonhuman primate model will bridge the rodent‐to‐human translational gap and enable detailed studies of the early‐life molecular determinants of AD pathogenesis.

CRISPR/Cas-based gene editing in therapeutic strategies for beta-thalassemia.

Beta-thalassemia (β-thalassemia) is an autosomal recessive disorder caused by point mutations, insertions, and deletions in the HBB gene cluster, resulting in the underproduction of β-globin chains. The most severe type may demonstrate complications including massive hepatosplenomegaly, bone deformities, and severe growth retardation in children. Treatments for β-thalassemia include blood transfusion, splenectomy, and allogeneic hematopoietic stem cell transplantation (HSCT). However, long-term blood transfusions require regular iron removal therapy. For allogeneic HSCT, human lymphocyte antigen (HLA)-matched donors are rarely available, and acute graft-versus-host disease (GVHD) may occur after the transplantation. Thus, these conventional treatments are facing significant challenges. In recent years, with the advent and advancement of CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) gene editing technology, precise genome editing has achieved encouraging successes in basic and clinical studies for treating various genetic disorders, including β-thalassemia. Target gene-edited autogeneic HSCT helps patients avoid graft rejection and GVHD, making it a promising curative therapy for transfusion-dependent β-thalassemia (TDT).In this review, we introduce the development and mechanisms of CRISPR/Cas9. Recent advances on feasible strategies of CRISPR/Cas9 targeting three globin genes (HBB, HBG, and HBA) and targeting cell selections for β-thalassemia therapy are highlighted. Current CRISPR-based clinical trials in the treatment of β-thalassemia are summarized, which are focused on γ-globin reactivation and fetal hemoglobin reproduction in hematopoietic stem cells. Lastly, the applications of other promising CRISPR-based technologies, such as base editing and prime editing, in treating β-thalassemia and the limitations of the CRISPR/Cas system in therapeutic applications are discussed.

Simultaneous CAS9 editing of cpSRP43, LHCA6, and LHCA7 in Picochlorum celeri lowers chlorophyll levels and improves biomass productivity.

High cellular pigment levels in dense microalgal cultures contribute to excess light absorption. To improve photosynthetic yields in the marine microalga Picochlorum celeri, CAS9 gene editing was used to target the molecular chaperone cpSRP43. Depigmented strains (>50% lower chlorophyll) were generated, with proteomics showing attenuated levels of most light harvesting complex (LHC) proteins. Gene editing generated two types of cpSRP43 transformants with distinct lower pigment phenotypes: (i) a transformant (Δsrp43) with both cpSRP43 diploid alleles modified to encode non-functional polypeptides and (ii) a transformant (STR30309) with a 3nt in-frame insertion in one allele at the CAS9 cut site (non-functional second allele), leading to expression of a modified cpSRP43. STR30309 has more chlorophyll than Δsrp43 but substantially less than wild type. To further decrease light absorption by photosystem I in STR30309, CAS9 editing was used to stack in disruptions of both LHCA6 and LHCA7 to generate STR30843, which has higher (5-24%) productivities relative to wild type in solar-simulating bioreactors. Maximal productivities required frequent partial harvests throughout the day. For STR30843, exemplary diel bioreactor yields of ~50 g m-2day-1 were attained. Our results demonstrate diel productivity gains in P.celeri by lowering pigment levels.

The HDAC2-MTA3 interaction induces nonsmall cell lung cancer cell migration and invasion by targeting c-Myc and cyclin D1.

Genome-wide association studies have identified numerous single-nucleotide polymorphisms (SNPs) associated with lung cancer; however, the functions of histone deacetylase 2 (HDAC2) rs13213007 and HDAC2 in nonsmall cell lung cancer (NSCLC) remain unclear. Here we identified HDAC2 rs13213007 as a risk SNP and showed that HDAC2 was upregulated in both peripheral blood mononuclear cells (PBMCs) and NSCLC tissues with the rs13213007 A/A genotype compared with those with the rs13213007 G/G or G/A genotype. Patient clinical data indicated strong associations between rs13213007 genotype and N classification. Immunohistochemical staining confirmed that higher expression of HDAC2 was associated with NSCLC progression. Furthermore, we generated 293T cells with the rs13213007 A/A genotype using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing. Chromatin immunoprecipitation sequencing followed by motif analysis showed that HDAC2 can bind to c-Myc in rs13213007 A/A 293T cells. Cell Counting Kit-8, colony formation, wound-healing, and Transwell assays revealed that HDAC2 upregulates c-Myc and cyclin D1 expression and promotes NSCLC cell proliferation, migration, and invasion. Co-immunoprecipitation, quantitative reverse transcription-polymerase chain reaction, and western blot analysis assays showed that MTA3 interacts with HDAC2, decreases HDAC2 expression, and rescues the migration and invasion abilities of NSCLC cells. Taken together, these findings identify HDAC2 as a potential therapeutic biomarker in NSCLC.

Siglec-9 is an inhibitory receptor on human mast cells in vitro

Mast cell activation is critical for the development of allergic diseases. Ligation of sialic acid-binding immunoglobin-like lectins (Siglecs), such as Siglec-6, -7, and -8 as well as CD33, have been shown to inhibit mast cell activation. Recent studies showed that human mast cells express Siglec-9, an inhibitory receptor also expressed by neutrophils, monocytes, macrophages, and dendritic cells. We aimed to characterize Siglec-9 expression and function in human mast cells invitro. We assessed the expression of Siglec-9 and Siglec-9 ligands on human mast cell lines and human primary mast cells by real-time quantitative PCR, flow cytometry, and confocal microscopy. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt the SIGLEC9 gene. We evaluated Siglec-9 inhibitory activity on mast cell function by using native Siglec-9 ligands, glycophorin A(GlycA), and high-molecular-weight hyaluronic acid, a monoclonal antibody against Siglec-9, and coengagement of Siglec-9 with the high-affinity receptor for IgE (FcεRI). Human mast cells express Siglec-9 and Siglec-9 ligands. SIGLEC9 gene disruption resulted in increased expression of activation markers at baseline and increased responsiveness to IgE-dependent and IgE-independent stimulation. Pretreatment with GlycA or high-molecular-weight hyaluronic acid followed by IgE-dependent or -independent stimulation had an inhibitory effect on mast cell degranulation. Coengagement of Siglec-9 with FcεRI in human mast cells resulted in reduced degranulation, arachidonic acid production, and chemokine release. Siglec-9 and its ligands play an important role in limiting human mast cell activation invitro.

Assessment of anti-HIV-1 guide RNA efficacy in cells containing the viral target sequence, corresponding gRNA, and CRISPR/Cas9.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing system has been shown to be effective at inhibiting human immunodeficiency virus type 1 (HIV-1). Studies have not consistently used a trackable dual reporter system to determine what cells received the Cas9/gRNA to determine the overall knockdown of HIV. Some studies have used stably transduced cells under drug selection to accomplish this goal. Here a two-color system was used that allows tracking of viral protein expression and which cells received the CRISPR/Cas9 system. These experiments ensured that each gRNA used was a perfect match to the intended target to remove this variable. The data showed that gRNAs targeting the transactivation response element (TAR) region or other highly conserved regions of the HIV-1 genome were effective at stopping viral gene expression, with multiple assays demonstrating greater than 95 percent reduction. Conversely, gRNAs targeting conserved sites of the 5' portion of the U3 region were largely ineffective, demonstrating that the location of edits in the long terminal repeat (LTR) matter with respect to function. In addition, it was observed that a gRNA targeting Tat was effective in a T-cell model of HIV-1 latency. Taken together, these studies demonstrated gRNAs designed to highly conserved functional regions have near 100% efficacy in vitro in cells known to have received the Cas9/gRNA pair.

Gene and cell therapy approaches for familial hypercholesterolemia: An update.

Familial hypercholesterolemia (FH) is a common autosomal codominant hereditary illness marked by the heightened risk of early atherosclerotic cardiovascular disease and high blood levels of low-density lipoprotein cholesterol (LDL-C). FH patients can have homozygous or heterozygous variants. This condition has been linked to variations in the genes for the LDL receptor (LDLR), apolipoprotein B, proprotein convertase subtilisin/Kexin 9 (PCSK9), and LDLR adaptor protein 1. Drugs such as statins, ezetimibe, and PCSK9 inhibitors are currently widely available, allowing for the theoretical normalization of plasma LDL-C levels mostly in patients with heterozygous FH. However, homozygous FH patients usually have a poor response to traditional lipid-lowering therapy and may have a poor prognosis at a young age. LDL apheresis and novel pharmacological therapies such as microsomal transfer protein inhibitors or anti-angiopoietin-like protein 3 monoclonal antibodies are extremely expensive and unavailable in most regions of the world. Therefore, the unmet need persists for these patients. In this review, we discuss the numerous gene delivery, gene editing, and stem cell manipulation techniques used in this study to correct FH-causing LDLR gene variations in vitro, ex vivo, and in vivo. Finally, we looked at a variety of studies that corrected genetic defects that caused FH using the ground-breaking clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology.

Generation of Genome-Edited Mice by Cytoplasmic Injection of CRISPR-Cas9 RNA.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR-Cas9) gene editing enables rapid production of genome-edited animals. The Cas9/guide RNA (gRNA) component can be introduced into zygotes in several ways. Here, we provide an instructional guide for the generation of knockout mice using cytoplasmic injection of in vitro transcribed Cas9 RNA and gRNA.

CRISPR-Cas9 gene editing and human diseases.

CRISPR/Cas-9 mediated genome editing has recently emerged as a potential and innovative technology in therapeutic development and biomedical research. Several recent studies have been performed to understand gene modification techniques in obtaining effective ex vivo results. Generally, the disease targets for gene correction will be in specific organs, so understanding the complete potential of genomic treatment methods is essential. From such a perspective, the present review revealed the significant importance of the CRISPR/ Cas9 delivery system. Both the promising gene-editing delivery systems, such as synthetic (non-viral) and viral vector systems are discussed in this review. In addition, this paper attempted to summarize the tissue-specific and organ-specific mRNA delivery systems that provide possible research information for future researchers. Further, the major challenges of the CRISPR/Cas9 system, such as off-target delivery, immunogenicity, and limited packaging, were also elucidated. Accordingly, this review illustrated a wide range of clinical applications associated with the efficient delivery of CRISPR/ Cas9 gene-editing. Moreover, this article emphasizes the role of the CRISPR/Cas9 system in treating Intra Cerebral haemorrhage (ICH), thereby suggesting future researchers to adopt more clinical trials on this breakthrough delivery system.

Two-Photon Calcium Imaging in the Brain of Aedes aegypti Mosquitoes.

Understanding the neural basis of mosquito behavior is critical for designing effective vector control strategies and can potentially shed new light on basic nervous system function. Because mosquitoes are a non-model species, however, functional studies of mosquito nervous systems have long been restricted to electrophysiological recording from peripheral sensory organs such as the antenna. This is now changing with the advent of CRISPR-Cas9 gene editing and the development of other powerful new genetic tools. Transgenic mosquitoes that carry genetically encoded calcium sensors, for example, open the door to optical recording of neural activity with two-photon calcium imaging. Compared with electrophysiology, calcium imaging permits continuous monitoring of neural activity from large populations of neurons, even deep in the brain. When combined with selective neural drivers, it also allows targeted recording from specific neuronal types. Here, we describe a calcium imaging protocol we use in our laboratory to study neural activity in the brain of Aedes aegypti mosquitoes.