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1.
Braz. J. Psychiatry (São Paulo, 1999, Impr.) ; 45(2): 137-145, Mar.-Apr. 2023. graf
Article in English | LILACS-Express | LILACS | ID: biblio-1439554

ABSTRACT

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.

2.
Rev. invest. clín ; 75(1): 13-28, Jan.-Feb. 2023. graf
Article in English | LILACS-Express | LILACS | ID: biblio-1450099

ABSTRACT

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.

3.
Chinese Journal of Experimental Ophthalmology ; (12): 925-930, 2023.
Article in Chinese | WPRIM | ID: wpr-990931

ABSTRACT

Several mutant genes for inherited retinal diseases have been identified, but effective treatments are still lacking.The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system can edit human genomic DNA by nonhomologous end joining or homology-directed repair, offering more possibilities for the treatment of hereditary retinal diseases.CRISPR/Cas9 not only can genetically correct patient-derived induced pluripotent stem cells (iPSCs) to observe their differentiation into retinal cells thereby, thereby exploring the pathogenesis of the disease and implementing cell therapy, but can also be delivered to the body via vectors and directly act on target cells to achieve in vivo gene editing.CRISPR/Cas9 gene editing technology in hereditary retinal diseases has been mainly used in retinitis pigmentosa, hereditary X-linked juvenile retinoschisis, and Leber congenital amaurosis 10, of which the in vitro application of CRISPR/Cas9 for Leber congenital amaurosis 10 has entered the clinical trial stage.In this paper, we reviewed the mechanism and key advances of CRISPR/Cas9 and provided an overview of gene editing in IRDs.

4.
International Journal of Biomedical Engineering ; (6): 144-150, 2023.
Article in Chinese | WPRIM | ID: wpr-989330

ABSTRACT

Cervical cancer is the fourth-ranked malignant tumor of female cancer in the world, and it seriously threatens women’s health. The main treatment options for patients with cervical cancer are surgery or concurrent chemoradiotherapy. With the development of medical research, researchers are committed to exploring more effective and specific treatment options in order to increase the treatment options for cervical cancer and improve the treatment effect. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is a method in which the Cas9 protein uses guide RNA (gRNA) to target the target gene and achieve precise editing of the target gene. At present, CRISPR/Cas9 technology has become a promising and powerful gene editing tool, a new and effective targeted therapy that has been applied in the treatment of various tumors. The research progress of CRISPR/Cas9 technology in the treatment of cervical cancer is mainly reviewed in terms of action targets, combination therapy strategies, and related drug resistance gene screening in order to provide new strategies for the treatment of cervical cancer.

5.
Journal of Southern Medical University ; (12): 516-526, 2023.
Article in Chinese | WPRIM | ID: wpr-986957

ABSTRACT

OBJECTIVE@#To establish a rapid detection and genotyping method for SARS-CoV-2 Omicron BA.4/5 variants using CRISPPR-Cas12a gene editing technology.@*METHODS@#We combined reverse transcription-polymerase chain reaction (RT-PCR) and CRISPR gene editing technology and designed a specific CRISPPR RNA (crRNA) with suboptimal protospacer adjacent motifs (PAM) for rapid detection and genotyping of SARS- CoV-2 Omicron BA.4/5 variants. The performance of this RT- PCR/ CRISPPR-Cas12a assay was evaluated using 43 clinical samples of patients infected by wild-type SARS-CoV-2 and the Alpha, Beta, Delta, Omicron BA. 1 and BA. 4/5 variants and 20 SARS- CoV- 2-negative clinical samples infected with 11 respiratory pathogens. With Sanger sequencing method as the gold standard, the specificity, sensitivity, concordance (Kappa) and area under the ROC curve (AUC) of RT-PCR/CRISPPR-Cas12a assay were calculated.@*RESULTS@#This assay was capable of rapid and specific detection of SARS- CoV-2 Omicron BA.4/5 variant within 30 min with the lowest detection limit of 10 copies/μL, and no cross-reaction was observed in SARS-CoV-2-negative clinical samples infected with 11 common respiratory pathogens. The two Omicron BA.4/5 specific crRNAs (crRNA-1 and crRNA-2) allowed the assay to accurately distinguish Omicron BA.4/5 from BA.1 sublineage and other major SARS-CoV-2 variants of concern. For detection of SARS-CoV-2 Omicron BA.4/5 variants, the sensitivity of the established assay using crRNA-1 and crRNA-2 was 97.83% and 100% with specificity of 100% and AUC of 0.998 and 1.000, respectively, and their concordance rate with Sanger sequencing method was 92.83% and 96.41%, respectively.@*CONCLUSION@#By combining RT-PCR and CRISPPR-Cas12a gene editing technology, we successfully developed a new method for rapid detection and identification of SARS-CoV-2 Omicron BA.4/5 variants with a high sensitivity, specificity and reproducibility, which allows rapid detection and genotyping of SARS- CoV-2 variants and monitoring of the emerging variants and their dissemination.


Subject(s)
Humans , COVID-19 , CRISPR-Cas Systems , Genotype , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , RNA , COVID-19 Testing
6.
Chinese Journal of Biologicals ; (12): 145-150+157, 2023.
Article in Chinese | WPRIM | ID: wpr-965608

ABSTRACT

@#Objective To knockout interferon alpha/beta receptor subunit 1(IFNAR1) gene in human colorectal adenocarcinoma cells Caco-2 using clustered regularly interspaced short palinmic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system to construct IFNAR1 knockout Caco-2 cell line.Methods The single guide RNA(sgRNA)sequence was designed to specifically recognize the exon region of IFNAR1 gene using CRISPR/Cas9 technology,and the LentiCRISPRv2-IFNAR1-sgRNA recombinant plasmid was constructed.Caco-2 cells were infected with the plasmid packaged by lentivirus and screened by puromycin resistance.The obtained monoclonal cell lines were cultured by limited dilution method,which were verified for the effect of IFNAR1 gene knockout by target gene sequencing and Western blot,and detected for the mRNA levels of CXC chemokine ligand 10(CXCL10)and interferon-stimulatd gene 20(ISG20)in IFNAR1knockout cells by adding exogenous IFNβ.Results Sequencing results of plasmid LentiCRISPRv2-IFNAR1-sgRNA showed that the insertion sites were all located at the sticky end of BsmBⅠenzyme digestion.Two IFNAR1 knockout monoclonal cell lines were obtained.The sequencing results showed that Caco-2-IFNAR1-KO1 had 5 bp deletion in the sixth exon of IFNAR1,and Caco-2-IFNAR1-KO2 had 18 bp deletion and 1 bp insertion in the seventh exon.Compared with wild-type Caco-2 cells,Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells showed no expression of IFNAR1 protein.Compared with no IFNβ stimulation,the mRNA levels of CXCL10 gene(t = 0.566 and 1.268 respectively,P>0.05)and ISG20 gene(t =1.522 and 1.733 respectively,P>0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells stimulated by 50 ng/mL IFNβ showed no significant increase.While compared with those of wild-type Caco-2 cells,the mRNA levels of CXCL10gene(t = 6.763 and 6.777 respectively,P<0.05)and ISG20 gene(t = 5.664 and 5.65 respectively,P<0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells decreased significantly under the stimulation of 50 ng/mL exogenous IFNβ.Conclusion Caco-2 cell line with IFNAR1 knockout was successfully constructed by using CRISPR/Cas9 technology,and the downstream molecules activated by IFNAR(interferon alpha/beta receptor)in this cell line were obviously inhibited,which provided a powerful tool for further exploration of the innate immune response and replication packaging mechanism of Caco-2 cells after virus infection.

7.
Organ Transplantation ; (6): 227-2023.
Article in Chinese | WPRIM | ID: wpr-965046

ABSTRACT

Heart transplantation is one of the most effective strategies to treat end-stage heart failure. Multiple challenges, such as difficulty in preservation of heart allograft, rejection and postoperative complications, emerge in heart allotransplantation. After decades of research and practice, most problems have been resolved. Nevertheless, the shortage of donor organs has become increasingly prominent. To alleviate the shortage of donor organs, artificial heart and heart xenotransplantation have captivated attention, and obtained significant progress in recent years. The application of artificial heart in clinical practice has significantly enhanced the survival rate of patients with end-stage heart failure, which is expected to become the standard treatment for end-stage heart failure. Heart xenotransplantation still faces many challenges, which is still far from clinical application. In this article, the history of heart transplantation, development of heart allotransplantation, use of artificial heart and research progress on heart xenotransplantation were reviewed, and the future development direction of heart transplantation was predicted.

8.
Chinese Journal of Biotechnology ; (12): 1363-1373, 2023.
Article in Chinese | WPRIM | ID: wpr-981143

ABSTRACT

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.


Subject(s)
CRISPR-Cas Systems/genetics , RNA/genetics , Bacteria/genetics , Gene Editing , Archaea
9.
Acta Pharmaceutica Sinica B ; (6): 2510-2543, 2023.
Article in English | WPRIM | ID: wpr-982869

ABSTRACT

CRISPR, as an emerging gene editing technology, has been widely used in multiple fields due to its convenient operation, less cost, high efficiency and precision. This robust and effective device has revolutionized the development of biomedical research at an unexpected speed in recent years. The development of intelligent and precise CRISPR delivery strategies in a controllable and safe manner is the prerequisite for translational clinical medicine in gene therapy field. In this review, the therapeutic application of CRISPR delivery and the translational potential of gene editing was firstly discussed. Critical obstacles for the delivery of CRISPR system in vivo and shortcomings of CRISPR system itself were also analyzed. Given that intelligent nanoparticles have demonstrated great potential on the delivery of CRISPR system, here we mainly focused on stimuli-responsive nanocarriers. We also summarized various strategies for CIRSPR-Cas9 system delivered by intelligent nanocarriers which would respond to different endogenous and exogenous signal stimulus. Moreover, new genome editors mediated by nanotherapeutic vectors for gene therapy were also discussed. Finally, we discussed future prospects of genome editing for existing nanocarriers in clinical settings.

10.
Journal of Zhejiang University. Science. B ; (12): 1-13, 2023.
Article in English | WPRIM | ID: wpr-982397

ABSTRACT

Biotechnology policies and regulations must be revised and updated to reflect the most recent advances in plant-breeding technology. New Plant Breeding Techniques (NPBT) such as gene editing have been applied to address the myriad of challenges in plant breeding, while the use of NPBT as emerging biotechnological tools raises legal and ethical concerns. This study aims to highlight how gene editing is operationalized in the existing literature and examine the critical issues of ethical and legal issues of gene editing for plant breeding. We carried out a systematic literature review (SLR) to provide the current states of ethical and legal discourses surrounding this topic. We also identified critical research priority areas and policy gaps that must be addressed when designing the future governance of gene editing in plant breeding.

11.
China Journal of Chinese Materia Medica ; (24): 2931-2939, 2023.
Article in Chinese | WPRIM | ID: wpr-981425

ABSTRACT

The U6 promoter is an important element driving sgRNA transcription in the CRISPR/Cas9 system. Seven PqU6 promo-ter sequences were cloned from the gDNA of Panax quinquefolium, and the transcriptional activation ability of the seven promoters was studied. In this study, seven PqU6 promoter sequences with a length of about 1 300 bp were cloned from the adventitious roots of P. quinquefolium cultivated for 5 weeks. Bioinformatics tools were used to analyze the sequence characteristics of PqU6 promoters, and the fusion expression vectors of GUS gene driven by PqU6-P were constructed. Tobacco leaves were transformed by Agrobacterium tumefaciens-mediated method for activity detection. The seven PqU6 promoters were truncated from the 5'-end to reach 283, 287, 279, 289, 295, 289, and 283 bp, respectively. The vectors for detection of promoter activity were constructed with GUS as a reported gene and used to transform P. quinquefolium callus and tobacco leaves. The results showed that seven PqU6 promoter sequences(PqU6-1P to PqU6-7P) were cloned from the gDNA of P. quinquefolium, with the length ranged from 1 246 bp to 1 308 bp. Sequence comparison results showed that the seven PqU6 promoter sequences and the AtU6-P promoter all had USE and TATA boxes, which are essential elements affecting the transcriptional activity of the U6 promoter. The results of GUS staining and enzyme activity test showed that all the seven PqU6 promoters had transcriptional activity. The PqU6-7P with a length of 1 269 bp had the highest transcriptional activity, 1.31 times that of the positive control P-35S. When the seven PqU6 promoters were truncated from the 5'-end(PqU6-1PA to PqU6-7PA), their transcriptional activities were different in tobacco leaves and P. quinquefolium callus. The transcriptional activity of PqU6-7PA promoter(283 bp) was 1.59 times that of AtU6-P promoter(292 bp) when the recipient material was P. quinquefolium callus. The findings provide more ideal endogenous U6 promoters for CRISPR/Cas9 technology in ginseng and other medicinal plants.


Subject(s)
Panax/genetics , Promoter Regions, Genetic , Agrobacterium tumefaciens/genetics , Computational Biology , Cloning, Molecular
12.
Chinese Journal of Biotechnology ; (12): 399-424, 2023.
Article in Chinese | WPRIM | ID: wpr-970382

ABSTRACT

The CRISPR-Cas9 system is composed of a clustered regularly interspaced short palindromic repeat (CRISPR) and its associated proteins, which are widely present in bacteria and archaea, serving as a specific immune protection against viral and phage secondary infections. CRISPR-Cas9 technology is the third generation of targeted genome editing technologies following zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). The CRISPR-Cas9 technology is now widely used in various fields. Firstly, this article introduces the generation, working mechanism and advantages of CRISPR-Cas9 technology; secondly, it reviews the applications of CRISPR-Cas9 technology in gene knockout, gene knock-in, gene regulation and genome in breeding and domestication of important food crops such as rice, wheat, maize, soybean and potato. Finally, the article summarizes the current problems and challenges encountered by CRISPR-Cas9 technology and prospects future development and application of CRISPR-Cas9 technology.


Subject(s)
Gene Editing , CRISPR-Cas Systems/genetics , Plant Breeding , Crops, Agricultural/genetics , Technology
13.
Chinese Journal of Biotechnology ; (12): 60-73, 2023.
Article in Chinese | WPRIM | ID: wpr-970359

ABSTRACT

Rapid and accurate detection technologies are crucial for disease prevention and control. In particular, the COVID-19 pandemic has posed a great threat to our society, highlighting the importance of rapid and highly sensitive detection techniques. In recent years, CRISPR/Cas-based gene editing technique has brought revolutionary advances in biotechnology. Due to its fast, accurate, sensitive, and cost-effective characteristics, the CRISPR-based nucleic acid detection technology is revolutionizing molecular diagnosis. CRISPR-based diagnostics has been applied in many fields, such as detection of infectious diseases, genetic diseases, cancer mutation, and food safety. This review summarized the advances in CRISPR-based nucleic acid detection systems and its applications. Perspectives on intelligent diagnostics with CRISPR-based nucleic acid detection and artificial intelligence were also provided.


Subject(s)
Humans , CRISPR-Cas Systems/genetics , COVID-19/genetics , Pandemics , Artificial Intelligence , Nucleic Acids
14.
Salud colect ; 19: e4462, 2023.
Article in Spanish | LILACS-Express | LILACS | ID: biblio-1530369

ABSTRACT

RESUMEN El creciente campo de la reproducción humana asistida ha alcanzado hitos inimaginables. Su continuo desarrollo y las innovaciones que genera, en ocasiones, plantean dilemas tanto éticos como jurídicos. El presente ensayo trata de exponer los cambios progresivos que se están viviendo en el ámbito del origen de la vida debido al desarrollo de nuevas opciones y estrategias en reproducción humana asistida. En primer lugar, se realiza una reflexión interdisciplinar desde la ciencia, la ética y el derecho, sobre la naturaleza humana y los cambios a los que la sociedad se enfrenta, en particular, desde la perspectiva española. En segundo lugar, recoge una breve aproximación en torno a las técnicas biomédicas presentes o futuras en el campo de la reproducción humana. Concluye sobre la necesidad de reflexionar ante el vertiginoso avance de la ciencia en materia de reproducción humana asistida.


ABSTRACT The growing field of assisted human reproduction has achieved unimaginable milestones. Its continuous development and the innovations it generates at times pose both ethical and legal dilemmas. This essay aims to elucidate the progressive changes occurring in the realm of the origin of life due to the development of new options and strategies in assisted human reproduction. First, it constructs an interdisciplinary reflection on human nature and the changes society faces from the perspectives of science, ethics, and law, particularly from the perspective of Spain. Second, it provides a brief overview of current or future biomedical techniques in the field of human reproduction. It concludes with a discussion of the need to reflect on the rapid advancement of science in assisted human reproduction.

15.
Estud. Psicol. (Campinas, Online) ; 40: e210091, 2023. graf
Article in English | LILACS, INDEXPSI | ID: biblio-1448240

ABSTRACT

Objective: This study analyzed the relation between the position of scientists on embryo editing and the different types of knowledge involved. Methods: A lexical analysis of 151 scientific articles in the PubMed and Web of Science databases was conducted using the IRAMUTEQ software. Results: The results showed that gene editing in embryos is presented in two argumentative branches: the first refers to the editing technique and its possibilities; the second discusses the impacts of these techniques on the public arena. The results demonstrate a consensus regarding the potential of editing; however, dilemmas about its effectiveness were also highlighted. Conclusion: The presence of ethical conflicts with embryo editing among the specialists was evidenced especially regarding the birth of genetically modified babies. Therefore, gene editing is marked by conflicts that are not limited only to biological contexts, but that encompasses different aspects of social life.


Objetivo: O objetivo deste trabalho foi analisar a relação entre o posicionamento dos cientistas sobre a edição de embriões e os diferentes tipos de conhecimento envolvidos nesses debates. Método: Utilizando o software IRAMUTEQ realizou-se uma análise lexical de 151 artigos científicos nas bases de dados PubMed e Web of Science. Resultados: Os resultados demonstraram que a edição genética de embriões se apresenta em dois blocos argumentativos: o primeiro se refere à técnica de edição e suas possibilidades e o segundo discute os impactos dessas técnicas na arena pública. Os achados demonstram consenso sobre as potencialidades da edição, contudo dilemas sobre a sua eficácia foram também destacados. Conclusão: Evidenciou-se a presença de embates éticos sobre a edição de embriões entre os especialistas em relação ao nascimento de bebês geneticamente modificados. Observou-se que a edição genética é marcada por conflitos que não se limitam apenas a contextos biológicos, mas que tangem diferentes aspectos da vida social.


Subject(s)
Bioethics , Embryo, Mammalian , Gene Editing , Social Representation
16.
Chinese Journal of Biotechnology ; (12): 1290-1303, 2023.
Article in Chinese | WPRIM | ID: wpr-981138

ABSTRACT

Live biotherapeutic products (LBPs) refer to the living bacteria derived from human body intestinal gut or in nature that can be used to treat the human disease. However, the naturally screened living bacteria have some disadvantages, such as deficient therapeutic effect and great divergence, which fall short of the personalized diagnosis and treatment needs. In recent years, with the development of synthetic biology, researchers have designed and constructed several engineered strains that can respond to external complex environmental signals, which speeded up the process of development and application of LBPs. Recombinant LBPs modified by gene editing can have therapeutic effect on specific diseases. Inherited metabolic disease is a type of disease that causes a series of clinical symptoms due to the genetic defect of some enzymes in the body, which may cause abnormal metabolism the corresponding metabolites. Therefore, the use of synthetic biology to design LBPs targeting specific defective enzymes will be promising for the treatment of inherited metabolic defects in the future. This review summarizes the clinic applications of LBPs and its potential for the treatment of inherited metabolic defects.


Subject(s)
Humans , Bacteria/genetics , Gene Editing , Metabolic Diseases/therapy
17.
Chinese Journal of Biotechnology ; (12): 1275-1289, 2023.
Article in Chinese | WPRIM | ID: wpr-981137

ABSTRACT

As human microbiome research advances, a large body of evidence shows that microorganisms are closely related to human health. Probiotics were discovered and used as foods or dietary supplements with health benefits in the last century. Microorganisms have shown broader application prospects in human health since the turn of the century, owing to the rapid development of technologies such as microbiome analysis, DNA synthesis and sequencing, and gene editing. In recent years, the concept of "next-generation probiotics" has been proposed as new drugs, and microorganisms are considered as "live biotherapeutic products (LBP)". In a nutshell, LBP is a living bacterial drug that can be used to prevent or treat certain human diseases and indications. Because of its distinct advantages, LBP has risen to the forefront of drug development research and has very broad development prospects. This review introduces the varieties and research advances on LBP from a biotechnology standpoint, followed by summarizing the challenges and opportunities for LBP clinical implementations, with the aim to facilitate LBP development.


Subject(s)
Humans , Probiotics , Dietary Supplements , Bacteria , Drug Development , Biotechnology
18.
Chinese Journal of Biologicals ; (12): 400-2023.
Article in Chinese | WPRIM | ID: wpr-976169

ABSTRACT

@#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.

19.
Journal of China Pharmaceutical University ; (6): 141-149, 2023.
Article in Chinese | WPRIM | ID: wpr-972905

ABSTRACT

@#Chimeric antigen receptor T-cell (CAR-T) immunotherapy has made a breakthrough in the clinical treatment of a variety of hematological tumors.However, the CAR-T cell products listed at China and abroad are all autologous CAR-T.Compared with autologous CAR-T treatment, universal CAR-T exhibits significant advantages, which could fulfill the treatment demand of more patients, but also displays high technical barriers.This paper reviews the universal CAR-T, clearly points out the two major challenges faced by the development of universal CAR-T, and then summarizes and analyzes the feasible solutions according to the mechanism causing the two major problems.This paper also summarizes domestic and foreign companies producing universal CAR-T and the latest clinical progress of their superior products, and then discusses the feasibility of the development strategy from another aspect, in order to provide ideas for developing a new generation of universal CAR-T cell therapy products.

20.
Chinese Medical Ethics ; (6): 401-407, 2023.
Article in Chinese | WPRIM | ID: wpr-1005721

ABSTRACT

Klara and the Sun is the first work published by Kazuo Ishiguro after he won the Nobel Prize for Literature. Its publication attracted widespread attention for he turned his attention to the gene-editing technology. By telling the story of Clara, an artificial friend (AF) designed to accompany "lifted" children, the author revealed the ethical problems caused by the application of gene-editing technology to "lifted" children. From the theoretical perspective of bioethics and its basic principles, this paper interpreted the ethical issues of gene-editing technology from three aspects that: "lifted" technology, "lifted" technology implementer, and "lifted" technology implementation consequence, and analyzed Kazuo Ishiguro’s technical ethics proposition.

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