Novelty in improvement of CAR T cell-based immunotherapy with the aid of CRISPR system

Abstract Introduction Chimeric Antigen Receptor (CAR) T cells have tremendous potentials for cancer treatment; however, various challenges impede their universal use. These restrictions include the poor function of T cells in tumor microenvironments, the shortage of tumor-specific antigens and, finally, the high cost and time-consuming process, as well as the poor scalability of the method. Creative gene-editing tools have addressed each of these limitations and introduced next generation products for cell therapy. The clustered regularly interspaced short palindromic repeats-associated endonuclease 9 (CRISPR/Cas9) system has triggered a revolution in biology fields, as it has a great capacity for genetic manipulation. Method In this review, we considered the latest development of CRISPR/Cas9 methods for the chimeric antigen receptor T cell (CAR T)-based immunotherapy. Results The ability of the CRISPR/Cas9 system to generate the universal CAR T cells and also potent T cells that are persistent against exhaustion and inhibition was explored. Conclusion: We explained CRISPR delivery methods, as well as addressing safety concerns related to the use of the CRISPR/Cas9 system and their potential solutions.

A Precise and Portable Detection System for Infectious Pathogens Based on CRISPR/Cas Technology / 分析化学

Nucleic acid-based molecular diagnostic methods are considered the gold standard for detecting infectious pathogens.However,when applied to portable or on-site rapid diagnostics,they still face various limitations and challenges,such as poor specificity,cumbersome operation,and portability difficulties.The CRISPR(Clustered regularly interspaced short palindromic repeats)/CRISPR-associated protein(Cas)-fluorescence detection method holds the potential to significantly enhance the specificity and signal-to-noise ratio of nucleic acid detection.In this study,we developed a portable grayscale reader detection system based on loop-mediated isothermal amplification(LAMP)-CRISPR/Cas.On one hand,in the presence of CRISPR RNA(crRNA),the CRISPR/Cas12a system was employed to achieve precise fluorescent detection of self-designed LAMP amplification reactions for influenza A and influenza B viruses.This further validated the high selectivity and versatility of the CRISPR/Cas system.On the other hand,the accompanying independently developed portable grayscale reader allowed for low-cost collection of fluorescence signals and high-reliability visual interpretation.At the end of the detection process,it directly provided positive or negative results.Practical sample analyses using this detection system have verified its reliability and utility,demonstrating that this system can achieve highly sensitive and highly specific portable analysis of influenza viruses.

Construction of Raji-Luc CD19 KO Lymphoma Cell Line Using CRISPR/Cas9 Technology / 现代检验医学杂志

Objective To construct Raji-Luc lymphoma cells with CD19 knockout using CRISPR/Cas9 technology and preliminarily validate their immune escape ability.Methods PB-CRISPR-CD19 small guide RNA(sgRNA)plasmids was constructed,the optimal sgRNA sequence was screened,and Raji-Luc cells with pCAG-PBase,PB-CD19 sgRNA,and PB-CRISPR-Cas9 were co-transfected.Stable knockout monoclonal cell lines were screened by flow sorting and limit dilution method and the knockout effect was verified through gene sequence testing.The expression of luciferase on the surface of the cell line was detected by microplate reader,CD19 CAR-T and CD38 CAR-T previously constructed in the laboratory were used as effector cells,and the immune escape ability of Raji-Luc CD19 KO cell line was verified by universal luciferase chemiluminescence method.Results The transfection efficiency of Raji-Luc CD19 KO cells prepared by electro transfection was high,and the knockout efficiency of the two monoclonal cells was more than 99%.There was no significant difference in luciferase expression compared to the original Raji-Luc cells,and CD19 CAR-T cells could not be activated to the kill them.Conclusion Successfully constructed Raji-Luc CD19 KO lymphoma cell line.

Research progress in SARS-CoV-2 nucleic acid detection based on microfluidic platforms / 医疗卫生装备

The detection principle of microfluidic microfluidic technology was introduced.The current research status of microfluidic platform-based SARS-CoV-2 nucleic acid detection technologies were reviewed such as reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR),digital PCR,isothermal amplification and clustered regularly interspaced palindromic repeats/CRISPR-associated protein.The deficiencies of microfluidic platform-based SARS-CoV-2 nucleic acid detection were analyzed.It's pointed out microfluidic platform-based SARS-CoV-2 nucleic acid detection had to be optimized and validated clinically in specialty,sensitivity,detection limit,reproducibility,informatization,quality control and reagent cost.[Chinese Medical Equipment Journal,2024,45(1):101-107]

Construction of Dmd Gene Mutant Mice and Phenotype Verification in Muscle and Immune Systems / 实验动物与比较医学

Objective The aim is to utilize CRISPR/Cas9 gene editing technology to construct Dmd gene mutant mice with a point mutation in exon 23 of the Dmd gene. Subsequently, the phenotypic changes of the mice in muscles and immune systems are analyzed and verified, providing an evaluation model for Duchenne muscular dystrophy and other related diseases.MethodsBased on the sequence characteristics of exon 23 of the Dmd gene, small guide RNA (sgRNA) was designed and synthesized. Cas9 mRNA, sgRNA fragments, and oligo donor DNA were microinjected into fertilized eggs of C57BL/6J mice. After transferring the fertilized eggs to surrogate mice, F0 generation mice were born. After mating with F0 generation mice, offspring mice were obtained, and Dmd gene positive mutant (DmdMu/+) mice were obtained after genotype identification. Male hemizygous DmdMu/+(DmdMu/Y) mice were selected for phenotype validation. The body weight of live 3- and 9-month-old mice were recorded. Muscle tension was evaluated through the grid test. Hearts and semitendinosus muscles were collected, and the histopathological changes were observed using HE staining. Further, the expression of Dmd protein in muscle tissue of 9-month-old mice was analyzed by Western blotting.An acute inflammation model was established in DmdMu/Y mice using lipopolysaccharide induction. Peripheral blood from the submandibular vein was collected, and the changes in the proportion of neutrophils and monocytes were detected by flow cytometry.Results The results of genome sequencing and Western blotting confirmed the successful construction of Dmd gene point mutant mice (DmdMu/+ mice). Dmd protein expression was not detected in skeletal muscle and myocardium of DmdMu/+ mice, and it was significantly reduced compared to wild-type C57BL/6J mice (P<0.05). Compared with wild-type mice of the same background, DmdMu/Y mice at 3 and 9 months of age showed significant weight loss (P<0.01) and decreased muscle tension (P<0.05). 9-month-old DmdMu/Y mice exhibited significant pathological changes in skeletal muscle and myocardium, including widening of intermuscular space. Under normal condition, compared with wild-type mice, the proportion of neutrophils and monocytes in the peripheral blood of 3-month-old DmdMu/Y mice was significantly lower than that of wild-type mice (P<0.01). After lipopolysaccharide stimulation, the proportion of neutrophils in peripheral blood of 3-month-old DmdMu/Y mice remained significantly lower compared to that of wild-type mice (P<0.01). The proportion of neutrophils in peripheral blood of 9-month-old DmdMu/Y mice significantly decreased after lipopolysaccharide induction (P<0.01), with a trend of change observed in monocytes between groups.Conclusion The successful construction of the Dmd gene mutant mouse model has confirmed the vital function of Dmd gene in maintaining normal muscle tissue morphology and muscle tone. It preliminarily indicated that Dmd gene deletion could significantly reduce the proportion of neutrophils in peripheral blood, offering a new perspective for the study of immune system alterations in Duchenne muscular dystrophy patients.

Application of CRISPR/Cas9 gene editing technology in the research of precision oncology / 中国肿瘤生物治疗杂志

@#[摘 要] CRISPR/Cas9基因编辑技术通过精准定位和修改基因序列，可以识别与细胞增殖、迁移、侵袭和化疗耐药性相关的基因，不仅为理解肿瘤发生发展的分子机制奠定了基础，还为实现肿瘤的精准治疗提供了一种方便、高效的方法。由于其具有低成本、高效率的优点，被广泛地应用于精准肿瘤学的基础和临床研究当中，包括用于探寻抗肿瘤药物耐药靶点、筛查驱动基因、优化CAR-T和TCR-T细胞，以及筛选肿瘤靶向基因等。目前，已开展了十余项项使用CRISPR/Cas9技术治疗肿瘤的临床试验，策略多为利用CRISPR/Cas9技术敲除T细胞中的免疫检查点基因后回输患者，以达到免疫激活的效果，大多数研究仍处于Ⅰ期和Ⅱ期阶段。管CRISPR/Cas9基因编辑技术在肿瘤研究与治疗领域展现出了巨大潜力，但仍需面对脱靶效应，以及永久编辑可能带来的弊端等瓶颈，其实际临床效用仍有待更多的深入研究和大规模临床试验的严格验证。

Construction and gene identification of myeloid specific Spi1 knockout mice / 安徽医科大学学报

Objective @#To construct myeloid specific Spi1 gene knockout mice and analyze their genotypes , so as to provide animal model basis for the study of pathological mechanism of diseases and drug targets .@*Methods @#According to the principle of CRISPR/Cas9 technology and C re/LoxP system , sgRNA and Donor vectors were de signed and constructed . The transcript of Exon 2 ( Exon 2) was used as the knockout region , and Loxp elements were placed on both sides of Exon 2 . Cas9 protein , sgRNA and Donor vector were mixed and microinj ected into the fertilized eggs of C57BL/6J mice , the fertilized eggs were transplanted into the uterus of C57BL/6J pregnant female mice , and F0 generation was obtained after 19 ~ 20 days . Positive F0 mice were mated with C57BL/6J mice to ob tain stable F1 Spi1 flox/ + mice . Spi1 flox/ + mice of F1 generation were selfed to obtain Spi1 flox/flox mice . Spi1 flox/flox mated with Lyz2-Cre + mice to obtain Spi1 flox/ + /Lyz2-Cre + mice , and then mated with Spi1 flox/flox , the Spi1 flox/flox/Lyz2-Cre + mice were myeloid specific Spi1 gene knockout ( KO) mice . Spi1 flox/flox/Lyz2-cre - mice were used as wild type (WT) mice . DNA of WT and KO mice was extracted , and the genotypes were identified by agarose gel electro phoresis after PCR amplification . Western blot was used to detect the expression of spleen focus forming virus proviral integration oncogene , Spi - 1 /purine rich box - 1(PU . 1) in immune cells of WT and KO mice .@*Results@#The results of PCR identification showed that the genotype of mice with only 220 bp amplified by flox primer was Spi1 flox/flox homozygote , and the genotype of mice with 700 bp amplified by Lyz2-Cre primer was Lyz2-Cre + . Western blot showed that compared with WT group , the protein PU . 1 was not expressed in bone marrow derived macropha ges (BMDMs ) and peritoneal macrophages (PM) in KO group (P < 0.01) . There was no significant difference of statistics in the expression level of PU . 1 in T cells between KO mice and WT mice . The results of PCR and West ern blot showed that myeloid specific Spi1 KO mice were successfully constructed . @*Conclusion @#The myeloid spe cific Spi1 gene KO mice are successfully constructed and identified , which provides animal model basis for further revealing the potential mechanism of PU . 1 inimmune regulation .

Breeding and genotyping of T lymphocyte-conditional Spi1 knockout mice / 安徽医科大学学报

Objective @#To breed and identify the T lymphocyte-conditional Spi1 knockout mice for the further in- vestgation of the specific role of Spi1-encoded protein PU. 1 . @*Methods @#The Lck-Cre mice were mated with Spi1 flox/flox mice to obtain Lck-Cre ×Spi1 flox/flox mice (T lymphocyte-specific Spi1 knockout mice) , and the genotype was determined by polymerase chain reaction (PCR) and agarose gel electrophoresis . Magnetic beads were used to sort out the splenic T lymphocytes , and the knockdown efficiency of PU. 1 in T cells was detected by Western blot , quantitative real-time PCR ( qPCR) and flow cytometry. @*Results @#The Lck-Cre ×Spi1 flox/flox mouse genotype was stably inherited . Compared with Spi1 flox/flox mice , the expression level of PU. 1 was significantly reduced in splenic T cells of Lck-Cre ×Spi1 flox/flox mice . @*Conclusion @#In this study , the T lymphocyte-specific Spi1 knockout mice was successfully constructed by applying Cre/LoxP system and CRISPR/Cas9 technology , which provided a reliable an- imal model for the subsequent experiments of the specific role of PU. 1 in T cell-related diseases .

Application of the CRISPR/Cas system in gene editing and nucleic acid detection of parasitic diseases: a review / 中国血吸虫病防治杂志

CRISPR/Cas system, an adaptive immune system with clustered regularly interspaced short palindromic repeats, may interfere with exogenous nucleic acids and protect prokaryotes from external damages, is an effective gene editing and nucleic acid detection tools. The CRISPR/Cas system has been widely applied in virology and bacteriology; however, there is relatively less knowledge about the application of the CRISPR/Cas system in parasitic diseases. The review summarizes the mechanisms of action of the CRISPR/Cas system and provides a comprehensive overview of their application in gene editing and nucleic acid detection of parasitic diseases, so as to provide insights into future studies on parasitic diseases.

Construction of HIF-1α gene knockout plasmid and functional verification in naked mole rats based on CRISPR/Cas9 system / 中国实验动物学报

Objective To construct plasmids and knock out HIF-1α gene expression in an naked mole rat skin fibroblasts(NSF)cell line using CRISPR/Cas9 genomic editing technology,to provide an in vitro cell model for studying the mechanism of hypoxia tolerance and the occurrence and development of hypoxia-related diseases in naked mole rats.Methods We designed four pairs of single guide RNA(sgRNA)sequences targeting exons 1～4 of the NSF HIF-1αgene and successfully constructed an expression plasmid.The plasmid with the optimal sgRNA was identified and transfected into 293T cells,and the supernatant was used for detecting the virus titer.Lentivirus particles carrying sgRNAs of HIF-1α were transfected into NSF cells which express Cas9 protein,based on a previous protocol.After transfection,fluorescence signals were observed under a fluorescence microscope,and HIF-1α expression in NSF cells was detected by Western Blot and T7 endonuclease 1(T7E1)analysis.Results Sanger sequencing showed that the designed sgRNA was successfully inserted into pX459 and pKLV2-U6-sgRNA2 vectors,demonstrating successful construction of a recombinant plasmid for transfection.T7E1 digestion successfully removed three bands and the target efficiency of sgRNA was 54％.Western Blot showed that the HIF-1α gene was successfully knocked out and its protein level was significantly reduced in NSF cells from naked mole rats(P=0.0019).There were no obvious morphological changes in HIF-1α-knockout cells under the microscope,and gene knockout had no obvious effect on cell proliferation.Conclusions We successfully constructed an HIF-1α-knockout cell line using CRISPR/Cas9 technology,to provide an experimental basis for further studies of the biological function of HIF-1α,as well as the mechanism of hypoxia tolerance in naked mole rats.The result also provide a theoretical foundation for the prevention and treatment of hypoxia-related diseases.

Application of CRISPR/Cas System-integrated Paper-based Analytical Devices for Rapid Detection of Foodborne Pathogens / 生物化学与生物物理进展

Foods can be contaminated with foodborne pathogens through a variety of pathways, including water, air and soil. Food safety events caused by foodborne pathogens show a serious impact on human health. However, due to the diversity of foodborne pathogens and the complexity of food matrices, the rapid detection of foodborne pathogens was difficult. The conventional microbial culture and physiological and biochemical identification can hardly meet the need of rapid detection of foodborne pathogens in the field. It is necessary to develop rapid detection technologies for foodborne pathogens. Clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein (Cas) are an adaptive immune systems of prokaryotes with specific recognition and cleavage of nucleic acid sequences, which shows good potential for development of nucleic acid detection and biosensing in the field. According to different forms of application, paper-based analytical devices can be categorized into test paper, lateral flow assay and microfluidic paper-based chips, etc. As a good simplicity and low-cost analytical testing tools, they show good prospects in the field of rapid testing. Therefore, the rapid and sensitive detection of foodborne pathogens can be realized by combining the efficient recognition ability of CRISPR/Cas system and the simplicity of paper-based analytical devices. In this paper, we briefly introduce an overview of the CRISPR/Cas system for nucleic acid detection, and this section focuses on an overview of the features and principles of the class 2 system, including types II, V and VI, which uses a single effector. The application of CRISPR/Cas system based test paper analysis, lateral flow assay and microfluidic paper-based chips for the detection of foodborne pathogens are highlighted in the paper, and finally the advantages, current challenges and future prospects of CRISPR/Cas system in combination with paper-based analytical devices to establish detection methods are discussed.

Phosphatidylinositol 3-kinases Play a Suppressive Role in Electrotaxis of Dictyostelium Cells Through Akt and ERK / 生物化学与生物物理进展

ObjectivePhosphatidylinositol 3 kinases (PI3Ks) play an important role in cell directional movement by regulating F-actin. However, the structure and function of PI3Ks are complex. The role of PI3Ks in cell electrotaxis is not fully understood. Therefore, in this study, the model organism Dictyostelium discoideum cells were used as experimental materials to explore the role of PI3K1 and PI3K2 in electrotaxis. MethodsFirstly, PI3K1 coding gene pikA knockout mutant and PI3K2 coding gene pikB knockout mutant were constructed by CRISPR/Cas9 system. Secondly, two mutants were placed in a DC electric field with a strength of 12 V/cm and the electrotaxis were analyzed. ResultsData analysis showed that the direction index of wild-type cells in DC electric field was (0.86±0.03), while the direction index of pikA- and pikB- mutants in DC electric field was (0.95±0.02) and (0.94±0.03), respectively. In addition, the average trajectory speed of wild-type cells in the electric field was (3.34±0.08) μm/min, while the average trajectory speed of pikA- and pikB- mutants were (4.85±0.20) μm/min and (5.48±0.15) μm/min, respectively. The t test showed that there were significant differences in the directedness index and speed between the mutant and wild type. Western blot results showed that both phosphorylated Akt and phosphorylated ERK were significantly increased in pikA- and pikB- mutants. ConclusionPI3K1 and PI3K2 may inhibit the electrotaxis of Dictyostelium discoideum cells by increasing the activity of Akt and ERK.

Novel approaches for HTLV-1 therapy: innovative applications of CRISPR-Cas9

ABSTRACT The human T-cell lymphotropic virus type 1 (HTLV-1) is a single-stranded positive-sense RNA virus that belongs to the Retroviridae family, genus Deltaretro, and infects approximately five to 10 million people worldwide. Although a significant number of individuals living with HTLV-1 remain asymptomatic throughout their lives, some develop one or more severe clinical conditions, such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a progressive and debilitating disease, and/or a subtype of non-Hodgkin's lymphoma with a more threatening course known as adult T-cell leukemia/lymphoma (ATLL). Moreover, current therapeutic options are limited and focus primarily on treating symptoms and controlling viral latency. CRISPR-Cas9 gene editing is proposed as a promising tool to address the intricate links associated with HTLV-1. By targeting or silencing key genes during initial infection and dysregulating immune signaling pathways, CRISPR-Cas9 offers potential intervention opportunities. In this review, we address the therapeutic potential of CRISPR-Cas9 gene editing, as well as examine the primary mechanisms involved in editing potential target genes and discuss the existing evidence in the current scientific literature.

Aplicaciones clínicas de la herramienta CRISPR-Cas / Clinical Applications of the CRISPR-Cas Tool

Resumen El desarrollo de tecnologías para la edición del genoma ha abierto la posibilidad de apuntar directamente y modificar secuencias genómicas en casi todo tipo de células eucariotas. La edición del genoma ha ampliado nuestra capacidad para dilucidar la contribución de la genética a las enfermedades al promover la creación de modelos celulares y animales más precisos de procesos patológicos y ha comenzado a mostrar su potencial en una variedad de campos, que van desde la investigación básica hasta la biotecnología aplicada y biomédica. Entre estas tecnologías, el uso de las repeticiones palindrómicas cortas agrupadas regularmente espaciadas ha acelerado, en gran medida, el progreso de la edición de genes desde el concepto hasta la práctica clínica, generando, además, interés debido, no solo a su precisión y eficiencia, sino también a la rapidez y a los costos necesarios para su implementación en comparación con otras tecnologías de edición genómica. En esta revisión se presenta información recabada de publicaciones indexadas en la base de datos PubMed que se encontraron mediante el uso de palabras claves asociadas con la tecnología y que se filtraron para retener solo aquellas con evidencias de avances clínicamente relevantes y que permiten demostrar algunas de las aplicaciones que tiene esta tecnología en la investigación, pronóstico y tratamiento de enfermedades genéticas, cardiovasculares, virales, entre otras; esto con el objetivo de dar a conocer la situación actual de los avances en aplicaciones clínicas de la herramienta CRISPR-Cas y fomentar aún más la investigación en esta tecnología, la cual, tal como se evidencia a lo largo de esta revisión, posee una gran versatilidad y un amplio rango de aplicaciones, lo que ofrece una enorme oportunidad en el campo de la medicina genómica, pero que, a su vez, requiere un mayor fomento en su investigación para mejorar la tecnología y acercarla aún más a consolidar aplicaciones clínicas de uso seguro, confiable y consistente.

Abstract The development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all types of eukaryotic cells. Genome editing has expanded our ability to elucidate the contribution of genetics to disease by promoting the creation of more precise cellular and animal models of disease processes and has begun to show its potential in a variety of fields, ranging from basic research to applied and biomedical biotechnology. Among these technologies, the use of clustered regularly spaced short palindromic repeats have greatly accelerated the progress of gene editing from concept to clinical practice, further generating interest due not only to its precision and efficiency, but also to the speed and costs required for its implementation compared to other genomic editing methods. This review presents information collected from indexed publications in the PubMed database that were found by using keywords associated with the technology and filtered to retain only those with evidence of clinically relevant advances that demonstrate some of the applications that this technology has in research, prognosis, and treatment of genetic, cardiovascular, and viral diseases, among others; this with the aim of show the current situation of advances in clinical applications of the CRISPR-Cas tool and further encourage research in this technology, which, as evidenced throughout this review, has a great versatility and a wide range of applications, which offers an enormous opportunity in the field of genomic medicine but which, in turn, requires greater support in its research to improve the technology and bring it even closer to consolidating clinical applications of safe, reliable and consistent use.

CRISPR/Cas9 genome editing approaches for psychiatric research

Currently, genome editing technologies, such as clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), are predominantly used to model genetic diseases. This genome editing system can correct point or frameshift mutations in risk genes. Here, we analyze and discuss the advantages of genome editing, its current applications, and the feasibility of the CRISPR/Cas9 system in research on psychiatric disorders. These disorders produce cognitive and behavioral alterations and their etiology is associated with polygenetic and environmental factors. CRISPR/Cas9 may reveal the biological mechanisms of psychiatric disorders at a basic research level, translating a suitable clinical approach for use in the diagnosis and treatment of psychiatric disorders. Genetic diagnosis and treatment for these disorders have not yet been fully established in psychiatry due to the limited understanding of their heterogeneity and polygenicity. We discuss the challenges and ethical issues in using CRISPR/Cas9 as a tool for diagnosis or gene therapy.

Editing the Human Genome with CRISPR/Cas: A Review of its Molecular Basis, Current Clinical Applications, and Bioethical Implications

ABSTRACT CRISPR/Cas genes evolved in prokaryotic organisms as a mechanism of defense designed to identify and destroy genetic material from threatening viruses. A breakthrough discovery is that CRISPR/Cas system can be used in eukaryotic cells to edit almost any desired gene. This comprehensive review addresses the most relevant work in the CRISPR/Cas field, including its history, molecular biology, gene editing capability, ongoing clinical trials, and bioethics. Although the science involved is complex, we intended to describe it in a concise manner that could be of interest to diverse readers, including anyone dedicated to the treatment of patients who could potentially benefit from gene editing, molecular biologists, and bioethicists. CRISPR/Cas has the potential to correct inherited diseases caused by single point mutations, to knock-in the promoter of a gene whose expression is highly desirable or knockout the gene coding for a deleterious protein. CRISPR/Cas technique can also be used to edit ex vivo immune cells and reinsert them in patients, improving their efficiency in attacking malignant cells, limiting the infectious potential of viruses or modulating xenotransplant rejection. Very important bioethical considerations on this topic include the need to internationally regulate its use by ad hoc expert committees and to limit its use until safety and bioethical issues are satisfactorily resolved.

CRISPR/Cas9-mediated endogenous TCR knockout enhances TCR-T cells targeted killing HPV16-positive cervical cancer cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：基于CRISPR/Cas9基因编辑技术制备无内源TCR的TCR-T细胞并鉴定其在体外杀伤HPV16阳性宫颈癌SiHa细胞的功能。方法：培养健康志愿者外周血CD8+ T细胞和Jurkat细胞，CRISPR/Cas9基因编辑技术敲除CD8+ T、Jurkat细胞的TCR基因，制备过表达转基因TCR的重组慢病毒，在敲除内源性TCR的CD8+ T和Jurkat细胞中用慢病毒过表达转基因TCR制备TCR-T细胞，多色FCM检测TCR-T细胞中TCR和CD3的表达水平，荧光素酶活性实验检测TCR-T细胞对HPV16阳性SiHa细胞的杀伤效率。结果：CRIPSR/Cas9基因编辑技术高效地敲除了外周血CD8+ T细胞和Jurkat细胞中的TRAC和TRBC基因，敲除效率分别为（81.4±4.5）%、（98.5±0.07）%，制备的无内源TCR的TCR-T细胞高效表达转基因TCR，在外周血CD8+ T和Jurkat细胞中表达率为（66.0±17.8）%、（97.3±2.6）%，敲除内源TRAC和TRBC基因有效增强CD8+ T和Jurkat细胞膜表达转基因TCR（均P<0.01），敲除内源TCR增强TCR-T细胞特异性杀伤HPV16阳性的SiHa细胞[（71.4±1.0）% vs （35.1±2.0）%，P<0.01）]。结论：无内源TCR的TCR-T细胞显著增强转基因TCR的表达和对HPV16阳性宫颈癌SiHa细胞的靶向杀伤能力，为提高TCR-T细胞的临床疗效提供了实验依据。

Construction of CRISPR/Cas9 system targeting mouse Tsc1 and Tsc2 gene and verification of targeting effectiveness / 中国生物制品学杂志

@#ObjectiveTo design and construct CRISPR/Cas9 gene editing system targeting Tsc1 and Tsc2 genes,and verify the effectiveness of gene editing at cellular level.MethodsThree sgRNA guide sequences were designed for mouse Tsc1 and Tsc2 genes respectively. The sgRNA expression vector was constructed and co-transfected with the Cas9 expression plasmid into mouse N2a cells. After the positive cells were obtained through drug screening,the DNA fragments at the targeting site were amplified by PCR,and the targeting efficiency was verified by TA clone sequencing.ResultsThe five targets of Tsc1-M-sgRNA2 and Tsc1-M-sgRNA3 of Tsc1 gene and Tsc2-M-sgRNA1,Tsc2-M-sgRNA2 and Tsc2-M-sgRNA3 of Tsc2 gene were all edited,and the editing efficiency was 40%,80%,30%,30% and 20%,respectively.ConclusionA CRISPR-Cas9 gene editing system with editing efficiency targeting mouse Tsc1 and Tsc2 genes was successfully constructed.

Advances in the RNA-targeting CRISPR-Cas systems / 生物工程学报

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated proteins) system is an adaptive immune system of bacteria and archaea against phages, plasmids and other exogenous genetic materials. The system uses a special RNA (CRISPR RNA, crRNA) guided endonuclease to cut the exogenous genetic materials complementary to crRNA, thus blocking the infection of exogenous nucleic acid. According to the composition of the effector complex, CRISPR-Cas system can be divided into two categories: class 1 (including type Ⅰ, Ⅳ, and Ⅲ) and class 2 (including type Ⅱ, Ⅴ, and Ⅵ). Several CRISPR-Cas systems have been found to have very strong ability to specifically target RNA editing, such as type Ⅵ CRISPR-Cas13 system and type Ⅲ CRISPR-Cas7-11 system. Recently, several systems have been widely used in the field of RNA editing, making them a powerful tool for gene editing. Understanding the composition, structure, molecular mechanism and potential application of RNA-targeting CRISPR-Cas systems will facilitate the mechanistic research of this system and provide new ideas for developing gene editing tools.

Generation of Mlk3 KO mice by CRISPR/Cas9 and its effect on blood pressure / 生物工程学报

To explore the effect of Mlk3 (mixed lineage kinase 3) deficiency on blood pressure, Mlk3 gene knockout (Mlk3KO) mice were generated. Activities of sgRNAs targeted Mlk3 gene were evaluated by T7 endonuclease I (T7E1) assay. CRISPR/Cas9 mRNA and sgRNA were obtained by in vitro transcription, microinjected into zygote, followed by transferring into a foster mother. Genotyping and DNA sequencing confirmed the deletion of Mlk3 gene. Real- time PCR (RT-PCR), Western blotting or immunofluorescence analysis showed that Mlk3KO mice had an undetectable expression of Mlk3 mRNA or Mlk3 protein. Mlk3KO mice exhibited an elevated systolic blood pressure compared with wild-type mice as measured by tail-cuff system. Immunohistochemistry and Western blotting analysis showed that the phosphorylation of MLC (myosin light chain) was significantly increased in aorta isolated from Mlk3KO mice. Together, Mlk3KO mice was successfully generated by CRISPR/Cas9 system. MLK3 functions in maintaining blood pressure homeostasis by regulating MLC phosphorylation. This study provides an animal model for exploring the mechanism by which Mlk3 protects against the development of hypertension and hypertensive cardiovascular remodeling.