Clustered Regularly Interspaced Short Palindromic repeats/CRISPR-associated Protein 9 Research Articles

Reproductive Characteristics and Suitability of Sterile dead end Knockout Nibe Croaker as a Recipient for Intraperitoneal Germ Cell Transplantation.

The use of sterile recipients is crucial for efficiently producing donor-derived offspring through surrogate broodstock technology for practical aquaculture applications. Although knockout (KO) of the dead end (dnd) gene has been used in previous studies as a sterilization method, it has not been reported in marine fish. In this study, nibe croaker was utilized as a model for marine teleosts that produce small pelagic eggs, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized to produce dnd KO fish. The F1 generation, which carried a nonsense mutation in the dnd gene, was produced by mating founder individuals with wild-type counterparts. Subsequently, the F2 generation was produced by mating the resulting males and females. Among the F2 generations, 24.0% consisted of homozygous KO individuals. Histological analysis revealed that primordial germ cells (PGCs) were present in homozygous KO individuals at 10days post-hatching (dph), similar to wild-type individuals. However, by 20 dph, PGCs were absent in KO individuals. Furthermore, no germ cells were observed in the gonads of both sexes of homozygous KO individuals at 6months old, which is the typical maturity age for wild-type individuals of both sexes. In addition, when cryopreserved donor nibe croaker testicular cells were transplanted, only donor-derived offspring were successfully obtained through the spontaneous mating of homozygous KO recipient parents. Results indicate that dnd KO nibe croaker lacks germ cells and can serve as promising recipients, producing only donor-derived gametes as surrogate broodstock.

An Efficient Vector-Based CRISPR/Cas9 System in Zebrafish Cell Line.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely applied in animals as an efficient genome editing tool. However, the technique is difficult to implement in fish cell lines partially due to the lack of efficient promoters to drive the expression of both sgRNA and the Cas9 protein within a single vector. In this study, it was indicated that the zebrafish U6 RNA polymerase III (ZFU6) promoter could efficiently induce tyrosinase (tyr) gene editing and lead to loss of retinal pigments when co-injection with Cas9 mRNA in zebrafish embryo. Furthermore, an optimized all-in-one vector for expression of the CRISPR/Cas9 system in the zebrafish fibroblast cell line (PAC2) was constructed by replacing the human U6 promoter with ZFU6 promoter, basing on the lentiCRISPRV2 system that widely applied in mammal cells. This new vector could successfully target the cellular communication network factor 2a (ctgfa) gene and demonstrated its function in the PAC2 cell. Notably, the vector could also be used to edit the endogenous EMX1 gene in the mammal 293T cell line, implying its wide application potential. In conclusion, we established a new gene editing tool for zebrafish cell line, which could be a useful in vitro platform for high-throughput analyzing gene function in fish.

[Prediction of CRISPR/Cas9 off-target activity using multi-scale convolutional neural network].

Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) is a new generation of gene editing technology, which relies on single guide RNA to identify specific gene sites and guide Cas9 nuclease to edit specific location in the genome. However, the off-target effect of this technology hampers its development. In recent years, several deep learning models have been developed for predicting the CRISPR/Cas9 off-target activity, which contributes to more efficient and safe gene editing and gene therapy. However, the prediction accuracy remains to be improved. In this paper, we proposed a multi-scale convolutional neural network-based method, designated as CnnCRISPR, for CRISPR/Cas9 off-target prediction. First, we used one-hot encoding method to encode the sgRNA-DNA sequence pair, followed by a bitwise or operation on the two binary matrices. Second, the encoded sequence was fed into the Inception-based network for training and evaluating. Third, the well-trained model was applied to evaluate the off-target situation of the sgRNA-DNA sequence pair. Experiments on public datasets showed CnnCRISPR outperforms existing deep learning-based methods, which provides an effective and feasible method for addressing the off-target problems.

Mitigating the Off-target Effects in CRISPR/Cas9-mediated Genetic Editing with Bioinformatic Technologies

The biological and clinical fields have recognized the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9) system as a precise and efficient tool for editing the genome. Despite its merits, the system poses crucial challenges, notably the off-target effects which could lead to unintended mutations - a substantial impediment for clinical applications that may potentially compromise the validity of research and the safety of therapeutic applications. Bioinformatics plays a pivotal role in mitigating this risk. Utilizing more refined bioinformatic tools and algorithms, researchers can reduce off-target mutations remarkably. These instruments, powered by machine learning and computational modelling, are able to predict off-target effects and provide aid for the design of more efficient sgRNA. Despite these advancements, it remains crucial to continue to focus on the improvement and assessment of such bioinformatics strategies. This review aims to holistically explore the mechanism and applications of CRISPR/Cas9 genome editing, its off-target effects, and the consequent impacts, along with the potential of bioinformatics techniques to identify off-target risks and facilitate sgRNA design. This review will also incorporate a clinical trial on HIV-1 treatment as a case study to highlight the potential of bioinformatics in devising solutions to mitigate the potential off-target effects of CRISPR/Cas9-mediated genetic editing.

Challenges and Opportunities of Gene Therapy in Cancer

Challenges and Opportunities of Gene Therapy in Cancer

Fine mapping and identification of two NtTOM2A homeologs responsible for tobacco mosaic virus replication in tobacco (Nicotiana tabacum L.)

BackgroundTobacco mosaic virus (TMV) is a widely distributed viral disease that threatens many vegetables and horticultural species. Using the resistance gene N which induces a hypersensitivity reaction, is a common strategy for controlling this disease in tobacco (Nicotiana tabacum L.). However, N gene-mediated resistance has its limitations, consequently, identifying resistance genes from resistant germplasms and developing resistant cultivars is an ideal strategy for controlling the damage caused by TMV.ResultsHere, we identified highly TMV-resistant tobacco germplasm, JT88, with markedly reduced viral accumulation following TMV infection. We mapped and cloned two tobamovirus multiplication protein 2A (TOM2A) homeologs responsible for TMV replication using an F2 population derived from a cross between the TMV-susceptible cultivar K326 and the TMV-resistant cultivar JT88. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated loss-of-function mutations of two NtTOM2A homeologs almost completely suppressed TMV replication; however, the single gene mutants showed symptoms similar to those of the wild type. Moreover, NtTOM2A natural mutations were rarely detected in 577 tobacco germplasms, and CRISPR/Cas9-mediated variation of NtTOM2A led to shortened plant height, these results indicating that the natural variations in NtTOM2A were rarely applied in tobacco breeding and the NtTOM2A maybe has an impact on growth and development.ConclusionsThe two NtTOM2A homeologs are functionally redundant and negatively regulate TMV resistance. These results deepen our understanding of the molecular mechanisms underlying TMV resistance in tobacco and provide important information for the potential application of NtTOM2A in TMV resistance breeding.

Nanotechnology‐based CRISPR/Cas9 delivery system for genome editing in cancer treatment

AbstractThe clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (CRISPR/Cas9) systems initiate a revolution in genome editing, which have a significant potential for treating cancer. A significant amount of research has been conducted regarding genetic modification using CRISPR/Cas9 systems, and 33 clinical trials using ex vivo or in vivo CRISPR/Cas9 gene editing techniques have been carried out to treat cancer. Despite its potential advantages, the main obstacle to convert CRISPR/Cas9 technology into clinical genome editing applications is the safe and efficient transport of genetic material owing to various extra‐ and intracellular biological hurdles. We outline the characteristics of three forms of CRISPR/Cas9 cargos, plasmids, mRNA/sgRNA, and ribonucleoprotein (RNP) complexes in this review. The recent in vivo nanotechnology‐based delivery techniques for these three categories to treat cancer are then reviewed. In the end, we outline the prerequisites for effective and secure in vivo CRISPR/Cas9 delivery in clinical contexts and discuss challenges with current nanocarriers. This review offers a thorough overview of the CRISPR/Cas9 nano‐delivery system for the treatment of cancer, serving as a resource for the design and building of CRISPR/Cas9 delivery systems and offering fresh perspectives on the treatment of tumors.

CRISPR/Cas9 Ribonucleoprotein-Mediated Mutagenesis in Sporisorium reilianum.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has become the state of the art for mutagenesis in filamentous fungi. Here, we describe a ribonucleoprotein complex (RNP)-mediated CRISPR/Cas9 for mutagenesis in Sporisorium reilianum. The efficiency of the method was tested in vitro with a cleavage assay as well as in vivo with a GFP-expressing S. reilianum strain. We applied this method to generate frameshift- and knock-out mutants in S. reilianum without a resistance marker by using an auto-replicating plasmid for selection. The RNP-mediated CRISPR/Cas9 increased the mutagenesis efficiency, can be applied for all kinds of mutations, and enables a marker-free genome editing in S. reilianum. Key features • First CRISPR/Cas9 application in S. reilianum. • Generation of S. reilianum mutants without genomic integration of resistance marker. • Allows the generation of multiple gene knockouts as well as deletion of large genomic regions.

Advances of CRISPR/Cas9 in cancer research and treatment

Cancer is a disease that is very difficult to cure and a lot of people in the world die because of cancer. In the past, the treatments of cancer could only depend on radiotherapy, chemotherapy and surgery of removing tumors. But these ways have some weaknesses such as harming the health of the body and relapse of cancer. Whereas recently, completely curing cancer has become possible through the advances of gene-editing technologies. Due to its great effectiveness, simplicity of usage, and other advantages, the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 (CRISPR/Cas9) system is a widely used gene editing technique today. But there are still many challenges that are applied to the body. For example, the CRISPR/Cas9 system has an off-target effect and there are no suitable carriers to deliver it to the area of the body. The resolution of these problems may be made possible by future developments in the CRISPR/Cas9 system. The use, restrictions, and potential uses of the CRISPR/Cas9 system in the treatment of cancer are examined in this research. In addition, this paper compares other treatments of cancer to summarize the advantages and disadvantages of different methods.

Brain-targeted delivery of CRISPR/Cas9 mediated glioblastoma therapeutics

Glioblastoma multiforme (GBM) is a life-threatening malignant tumor of the central nervous system, for which there is currently no effective treatment. Its low survival rate has been interpreted as a result of its high proliferation rate, resistance to apoptosis, and the ability to create a microenvironment conducive to tumor growth. Recently, a powerful and accurate gene-editing tool, Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Protein 9 (CRISPR/Cas9), has high potential in various scientific fields. Such technologies can manipulate cellular mechanisms and defective genes that lead to the progression of many serious diseases such as cancer. One of the major barriers to the application of this technique is the development of a delivery method to diffuse CRISPR/Cas9 efficiently and accurately to the target location in brain. Most existing delivery methods are failed to be translated into clinical result due to the lack of promising safety and efficiency. Thus, I will introduce several strategies here that can be used potentially for CRISPR-Cas9 system delivery in GBM treatment including the principle, advantages, limitations, and latest developments of these systems. This review is composed to provide a concise summary for future researchers to understand the current challenges and approaches in CRISPR/Cas9 mediated GBM therapeutics delivery.

CRISPR/Cas9-Mediated Genome Editing in Cancer Therapy.

The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, an RNA-based adaptive immune system found in bacteria and archaea, has catalyzed the development and application of a new generation of gene editing tools. Numerous studies have shown that this system can precisely target a wide range of human genes, including those associated with diseases such as cancer. In cancer research, the intricate genetic mutations in tumors have promoted extensive utilization of the CRISPR/Cas9 system due to its efficient and accurate gene editing capabilities. This includes improvements in Chimeric Antigen Receptor (CAR)-T-cell therapy, the establishment of tumor models, and gene and drug target screening. Such progress has propelled the investigation of cancer molecular mechanisms and the advancement of precision medicine. However, the therapeutic potential of genome editing remains underexplored, and lingering challenges could elevate the risk of additional genetic mutations. Here, we elucidate the fundamental principles of CRISPR/Cas9 gene editing and its practical applications in tumor research. We also briefly discuss the primary challenges faced by CRISPR technology and existing solutions, intending to enhance the efficacy of this gene editing therapy and shed light on the underlying mechanisms of tumors.

Engineering Drought Tolerance in Crops Using CRISPR Cas systems

Drought stress is one of the most considerable threats to global agricultural food security, causing yield losses worldwide. Therefore, the search for effective genetic and molecular methods for developing cultivars that are tolerant or resistant to harsh environments has been more intensive over the last decades. Apart from time-consuming conventional breeding techniques, biotechnologists are now investigating modern genome editing tools for engineering tolerance and resistance to various biotic and abiotic stresses in crops. Various genetic engineering techniques such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) were developed based on the discovery of the DNA structure. However, these methods have limitations, with ZFNs being prone to errors due to their limited base pair recognition, and TALENs requiring a complex protein engineering process and struggling to cleave methylated DNA. In recent years, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) and its alternatives have gained popularity in plant biotechnology. Out of the genome editing techniques mentioned earlier, CRISPR/Cas9 is becoming more popular because it's faster and easier to use. Given that drought is now a significant threat to global agriculture due to the drying of arable lands, this review focuses on how we can use CRISPR genome editing to enhance crop tolerance to drought stress and explores its future potential.

NAC transcription factor SlNOR-like1 plays a dual regulatory role in tomato fruit cuticle formation.

The plant cuticle is an important protective barrier on the plant surface, constructed mainly by polymerized cutin matrix and a complex wax mixture. Although the pathway of plant cuticle biosynthesis has been clarified, knowledge of the transcriptional regulation network underlying fruit cuticle formation remains limited. In the present work, we discovered that tomato fruits of the NAC transcription factor SlNOR-like1 knockout mutants (nor-like1) produced by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9] displayed reduced cutin deposition and cuticle thickness, with a microcracking phenotype, while wax accumulation was promoted. Further research revealed that SlNOR-like1 promotes cutin deposition by binding to the promoters of glycerol-3-phosphate acyltransferase6 (SlGPAT6; a key gene for cutin monomer formation) and CUTIN DEFICIENT2 (SlCD2; a positive regulator of cutin production) to activate their expression. Meanwhile, SlNOR-like1 inhibits wax accumulation, acting as a transcriptional repressor by targeting wax biosynthesis, and transport-related genes 3-ketoacyl-CoA synthase1 (SlKCS1), ECERIFERUM 1-2 (SlCER1-2), SlWAX2, and glycosylphosphatidylinositol-anchored lipid transfer protein 1-like (SlLTPG1-like). In conclusion, SlNOR-like1 executes a dual regulatory effect on tomato fruit cuticle development. Our results provide a new model for the transcriptional regulation of fruit cuticle formation.

Heterologous overproduction of oviedomycin by refactoring biosynthetic gene cluster and metabolic engineering of host strain Streptomyces coelicolor

BackgroundOviedomycin is one among several polyketides known for their potential as anticancer agents. The biosynthetic gene cluster (BGC) for oviedomycin is primarily found in Streptomyces antibioticus. However, because this BGC is usually inactive under normal laboratory conditions, it is necessary to employ systematic metabolic engineering methods, such as heterologous expression, refactoring of BGCs, and optimization of precursor biosynthesis, to allow efficient production of these compounds.ResultsOviedomycin BGC was captured from the genome of Streptomyces antibioticus by a newly constructed plasmid, pCBA, and conjugated into the heterologous strain, S. coelicolor M1152. To increase the production of oviedomycin, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized in an in vitro setting to refactor the native promoters within the ovm BGC. The target promoters of refactoring were selected based on examination of factors such as transcription levels and metabolite profiling. Furthermore, genome-scale metabolic simulation was applied to find overexpression targets that could enhance the biosynthesis of precursors or cofactors related to oviedomycin production. The combined approach led to a significant increase in oviedomycin production, reaching up to 670 mg/L, which is the highest titer reported to date. This demonstrates the potential of the approach undertaken in this study.ConclusionsThe metabolic engineering approach used in this study led to the successful production of a valuable polyketide, oviedomycin, via BGC cloning, promoter refactoring, and gene manipulation of host metabolism aided by genome-scale metabolic simulation. This approach can be also useful for the efficient production of other secondary molecules encoded by ‘silent’ BGCs.

Altered Peripheral Blood Gene Expression in Childhood Cancer Survivors With Anthracycline-Induced Cardiomyopathy - A COG-ALTE03N1 Report.

Background Anthracycline-induced cardiomyopathy is a leading cause of premature death in childhood cancer survivors, presenting a need to understand the underlying pathogenesis. We sought to examine differential blood-based mRNA expression profiles in anthracycline-exposed childhood cancer survivors with and without cardiomyopathy. Methods and Results We designed a matched case-control study (Children's Oncology Group-ALTE03N1) with mRNA sequencing on total RNA from peripheral blood in 40 anthracycline-exposed survivors with cardiomyopathy (cases) and 64 matched survivors without (controls). DESeq2 identified differentially expressed genes. Ingenuity Pathway Analyses (IPA) and Gene Set Enrichment Analyses determined the potential roles of altered genes in biological pathways. Functional validation was performed by gene knockout in human-induced pluripotent stem cell-derived cardiomyocytes using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) technology. Median age at primary cancer diagnosis for cases and controls was 8.2 and 9.7 years, respectively. Thirty-six differentially expressed genes with fold change ≥±2 were identified; 35 were upregulated. IPA identified "hepatic fibrosis" and "iron homeostasis" pathways to be significantly modulated by differentially expressed genes, including toxicology functions of myocardial infarction, cardiac damage, and cardiac dilation. Leading edge analysis from Gene Set Enrichment Analyses identified lactate dehydrogenase A (LDHA) and cluster of differentiation 36 (CD36) genes to be significantly upregulated in cases. Interleukin 1 receptor type 1, 2 (IL1R1, IL1R2), and matrix metalloproteinase 8, 9 (MMP8, MMP9) appeared in multiple canonical pathways. LDHA-knockout human-induced pluripotent stem cell-derived cardiomyocytes showed increased sensitivity to doxorubicin. Conclusions We identified differential mRNA expression profiles in peripheral blood of anthracycline-exposed childhood cancer survivors with and without cardiomyopathy. Upregulation of LDHA and CD36 genes suggests metabolic perturbations in a failing heart. Dysregulation of proinflammatory cytokine receptors IL1R1 and IL1R2 and matrix metalloproteinases, MMP8 and MMP9 indicates structural remodeling that accompanies the clinical manifestation of symptomatic cardiotoxicity.

Benchmarking deep learning methods for predicting CRISPR/Cas9 sgRNA on- and off-target activities.

In silico design of single guide RNA (sgRNA) plays a critical role in clustered regularly interspaced, short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. Continuous efforts are aimed at improving sgRNA design with efficient on-target activity and reduced off-target mutations. In the last 5years, an increasing number of deep learning-based methods have achieved breakthrough performance in predicting sgRNA on- and off-target activities. Nevertheless, it is worthwhile to systematically evaluate these methods for their predictive abilities. In this review, we conducted a systematic survey on the progress in prediction of on- and off-target editing. We investigated the performances of 10 mainstream deep learning-based on-target predictors using nine public datasets with different sample sizes. We found that in most scenarios, these methods showed superior predictive power on large- and medium-scale datasets than on small-scale datasets. In addition, we performed unbiased experiments to provide in-depth comparison of eight representative approaches for off-target prediction on 12 publicly available datasets with various imbalanced ratios of positive/negative samples. Most methods showed excellent performance on balanced datasets but have much room for improvement on moderate- and severe-imbalanced datasets. This study provides comprehensive perspectives on CRISPR/Cas9 sgRNA on- and off-target activity prediction and improvement for method development.

Metallothionein Gene Deficiency Facilitates the Differentiation of C2C12 Myoblasts into Slow-Twitch Myotubes.

Metallothionein (MT) 1 and 2 are ubiquitously expressed cysteine-rich, low molecular weight proteins. MT expression is upregulated in skeletal muscle during aging. MTs also play role in multiple types of skeletal muscle atrophy. Meanwhile, it has been reported that MT1 and MT2 gene deficiency increases myogenesis in MT knockout (MTKO) mice. However, little is known about the effect of MTs on muscle formation and atrophy. In this study, we investigated the effect of MT1 and MT2 gene knock-out using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) system in an in vitro skeletal muscle differentiation model (C2C12 cell line). MT deficiency promoted myogenic differentiation and myotube formation in C2C12 cells. Muscle-specific transcription factors MyoD and myogenin were found to be upregulated at the late stage of myotube differentiation in MTKO cells. Furthermore, the fast-twitch myosin heavy chain (MyHC) protein expression was similar in MTKO and mock-transfected myotubes, but slow-MyHC expression was higher in MTKO cells than in mock cells. The MT gene deletion did not affect the number of fast MyHC-positive myotubes but increased the number of slow MyHC-positive myotubes. Treatment with the antioxidant N-acetylcysteine (NAC) inhibited the increase in the number of slow MyHC-positive myotubes as well as slow-MyHC expression in MTKO cells. In contrast, NAC treatment did not alter the number of fast MyHC-positive myotubes or the expression of fast-MyHC in MTKO cells. These results suggest that the antioxidant effects of MTs may be involved in slow-twitch myofiber formation in skeletal muscle.

CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma

BackgroundGlioblastoma (GBM) is the most common malignant brain tumor and has “immunologically cold” features. Changing GBM to an “immunologically hot” tumor requires a strong trigger that induces initial immune responses in GBM. Allogeneic natural killer cells (NKCs) have gained considerable attention as promising immunotherapeutic tools against cancer, where gene-edited NKCs would result in effective anti-cancer treatment. The present study focused on the immune checkpoint molecule cytokine-inducible SH2-containing protein (CISH, or CIS) as a critical negative regulator in NKCs.MethodsThe GBM tumor environment featured with immunological aspect was analyzed with Cancer immunogram and GlioVis. We generated human primary CIS-deleted NKCs (NK dCIS) using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) with single guide RNA targeting genome sites on CIS coding exons. The genome-edited NKCs underwent microarray with differential expression analysis and gene set enrichment analysis (GSEA). The anti-GBM activity of the genome-edited NKCs was evaluated by apoptosis induction effects against allogeneic GBM cells and spheroids. We further detected in vivo antitumor effects using xenograft brain tumor mice.ResultsWe successfully induced human CIS-deleted NKCs (NK dCIS) by combining our specific human NKC expansion method available for clinical application and genome editing technology. CIS gene-specific guide RNA/Cas9 protein complex suppressed CIS expression in the expanded NKCs with high expansion efficacy. Comprehensive gene expression analysis demonstrated increased expression of 265 genes and decreased expression of 86 genes in the NK dCIS. Gene set enrichment analysis revealed that the enriched genes were involved in NKC effector functions. Functional analysis revealed that the NK dCIS had increased interferon (IFN)ɤ and tumor necrosis factor (TNF) production. CIS deletion enhanced NKC-mediated apoptosis induction against allogeneic GBM cells and spheroids. Intracranial administration of the allogeneic NKCs prolonged the overall survival of xenograft brain tumor mice. Furthermore, the NK dCIS extended the overall survival of the mice.ConclusionThe findings demonstrated the successful induction of human primary NK dCIS with CRISPR/Cas9 with efficient expansion. CIS deletion enhanced the NKC-mediated anti-tumor effects in allogeneic GBM and could be a promising immunotherapeutic alternative for patients with GBM.

Hypoimmunogenic Human Pluripotent Stem Cells as a Powerful Tool for Liver Regenerative Medicine.

Induced pluripotent stem cells (iPSC) have huge potential as cell therapy for various diseases, given their potential for unlimited self-renewal and capability to differentiate into a wide range of cell types. Although autologous iPSCs represents the ideal source for patient-tailored regenerative medicine, the high costs of the extensive and time-consuming production process and the impracticability for treating acute conditions hinder their use for broad applications. An allogeneic iPSC-based strategy may overcome these issues, but it carries the risk of triggering an immune response. So far, several approaches based on genome-editing techniques to silence human leukocyte antigen class I (HLA-I) or II (HLA-II) expression have been explored to overcome the immune rejection of allogeneic iPSCs. In this study, we employed the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system to delete the β2-Microglobulin (B2M) and the Class II Major Histocompatibility Complex Transactivator (CIITA) genes, essential for the correct surface expression of HLA-I and HLA-II proteins. The resulting hypoimmunogenic iPSC line has a normal karyotype, expresses the pluripotency stem cell markers, and is capable of differentiating into the three embryonic germ layers. Furthermore, we showed that it specifically retains the ability to differentiate towards different liver cells, such as endothelial-like cells, hepatocyte-like cells, and hepatic stellate-like cells. Our results indicate that hypoimmunogenic iPSCs could give a new cost-effective and off-the-shelf opportunity for cell therapy in liver diseases.

THE PROSPECTS AND CHALLENGES OF CRISPR/CAS9 GENE EDITING IN CANCER THERAPY: A LITERATURE REVIEW

Relevance: Cancer remains one of the leading causes of death in Kazakhstan, and CRISPR/Cas9 offers possible solutions to treat it. Clustered, regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is a system bacteria use to cleave foreign in-vaders. This system has been considered promising for cancer therapeutics by allowing researchers to edit cancer cell genes.
 The system requires more trials, so it is essential to raise awareness of this technique for stu-dents and potential investors and highlight the current challenges that could be research opportuni-ties for researchers.
 The study aimed to analyze and provide up-to-date information from reputable scientific journals on the current use of the CRISPR/Cas9 system in cancer therapeutics for medical students and researchers. This research paper also highlights the challenges associated with implementing CRISPR/Cas9 in clinical settings for cancer therapeutics.
 Methods: The scientific literature and databases (PubMed and the Nature Journal) were searched and analyzed using the CRISPR/Cas9 system in cancer therapy.
 Results: The results of this research indicate that scientists should focus on improving the types and structure of the Cas protein as well as the delivery methods, including the non-viral deliv-ery methods (liposome-based particles, hybrid vectors, gold nanoparticles, and extracellular vesicles) to contribute to improving the current status of cancer therapeutics.
 Conclusion: CRISPR/Cas9 is an important technique that is still fraught with challenges and should be turned into research opportunities. The current challenges include the form and structure of the Cas nuclease, the types of engineering (in vivo vs. ex vivo), and the varieties of delivery methods. Each delivery method type has pros and cons and requires further research. In particular, future studies should focus on non-viral vectors, such as liposome-based particles, extracellular vesi-cles, hybrid vesicles, and gold nanoparticles.