Research on the Ethical Supervision of Human Gene Editing Technology: Based on the Perspective of Game Theory / 中国医学伦理学

In view of the current norms and demands of human gene editing technology at home and abroad, the paper explained that the regulatory difficulties faced by human gene editing technology were due to the conflict between economic interests and moral bottom line by constructing a game model in a hypothetical way. On this basis, the ideas of the supervision mode of human gene editing technology were put forward: establish unified international standards based on the country as the main body, enact more stringent and effective laws, to jointly deal with the behavior of genetic manipulation of human gametes, zygotes and embryos for the purpose of reproductive, and ensure the normalization and legalization of gene editing technology to avoid technology abuse.

Advances in corneal stromal regeneration and repair / 国际眼科杂志(Guoji Yanke Zazhi)

Corneal stroma is a significant part of the cornea and plays a significant role in the eye's refractive system. Although corneal transplantation is now the most effective treatment for corneal stromal disease, its advancement has been constrained by a shortage of donors, the need for prolonged immunosuppressive medicine to prevent rejection, and low graft survival rates. An alternate strategy is to use the corneal stroma's natural capacity for regeneration to create the ideal conditions for the collagenous extracellular matrix of the stroma to self-renew. However, it is challenging to replicate the intricate ultrastructure of the corneal stroma in vitro. Regenerative medicine has so been used to address these issues. These approaches refer to numerous disciplines, including stem cell-induced differentiation, tissue engineering and gene editing. This article provides potential directions for the future clinical applications of corneal stromal regeneration and repair while summarizing pertinent techniques, research progress, and issues.

Logic-gated tumor-microenvironment nanoamplifier enables targeted delivery of CRISPR/Cas9 for multimodal cancer therapy

Recent innovations in nanomaterials inspire abundant novel tumor-targeting CRISPR-based gene therapies. However, the therapeutic efficiency of traditional targeted nanotherapeutic strategies is limited by that the biomarkers vary in a spatiotemporal-dependent manner with tumor progression. Here, we propose a self-amplifying logic-gated gene editing strategy for gene/H2O2-mediated/starvation multimodal cancer therapy. In this approach, a hypoxia-degradable covalent-organic framework (COF) is synthesized to coat a-ZIF-8 in which glucose oxidase (GOx) and CRISPR system are packaged. To intensify intracellular redox dyshomeostasis, DNAzymes which can cleave catalase mRNA are loaded as well. When the nanosystem gets into the tumor, the weakly acidic and hypoxic microenvironment degrades the ZIF-8@COF to activate GOx, which amplifies intracellular H+ and hypoxia, accelerating the nanocarrier degradation to guarantee available CRISPR plasmid and GOx release in target cells. These tandem reactions deplete glucose and oxygen, leading to logic-gated-triggered gene editing as well as synergistic gene/H2O2-mediated/starvation therapy. Overall, this approach highlights the biocomputing-based CRISPR delivery and underscores the great potential of precise cancer therapy.

Gene therapy strategies and prospects for neurofibromatosis type 1 / 中国修复重建外科杂志

OBJECTIVE@#To summarize the gene therapy strategies for neurofibromatosis type 1 (NF1) and related research progress.@*METHODS@#The recent literature on gene therapy for NF1 at home and abroad was reviewed. The structure and function of the NF1 gene and its mutations were analyzed, and the current status as well as future prospects of the transgenic therapy and gene editing strategies were summarized.@*RESULTS@#NF1 is an autosomal dominantly inherited tumor predisposition syndrome caused by mutations in the NF1 tumor suppressor gene, which impair the function of the neurofibromin and lead to the disease. It has complex clinical manifestations and is not yet curable. Gene therapy strategies for NF1 are still in the research and development stage. Existing studies on the transgenic therapy for NF1 have mainly focused on the construction and expression of the GTPase-activating protein-related domain in cells that lack of functional neurofibromin, confirming the feasibility of the transgenic therapy for NF1. Future research may focus on split adeno-associated virus (AAV) gene delivery, oversized AAV gene delivery, and the development of new vectors for targeted delivery of full-length NF1 cDNA. In addition, the gene editing tools of the new generation have great potential to treat monogenic genetic diseases such as NF1, but need to be further validated in terms of efficiency and safety.@*CONCLUSION@#Gene therapy, including both the transgenic therapy and gene editing, is expected to become an important new therapeutic approach for NF1 patients.

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.

Application of CRISPR-Cas9 gene editing technology in crop breeding / 生物工程学报

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.

CRISPR-based molecular diagnostics: a review / 生物工程学报

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.

Construction of interferon alpha/beta receptor subunit 1 gene knockout Caco-2 cell line based on CRISPR/Cas9 system / 中国生物制品学杂志

@#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.

The past, present and future of heart transplantation / 器官移植

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.

Engineering and application of Komagataella phaffii as a cell factory / 生物工程学报

Nowadays, engineered Komagataella phaffii plays an important role in the biosynthesis of small molecule metabolites and protein products, showing great potential and value in industrial productions. With the development and application of new editing tools such as CRISPR/Cas9, it has become possible to engineer K. phaffii into a cell factory with high polygenic efficiency. Here, the genetic manipulation techniques and objectives for engineering K. phaffii are first summarized. Secondly, the applications of engineered K. phaffii as a cell factory are introduced. Meanwhile, the advantages as well as disadvantages of using engineered K. phaffii as a cell factory are discussed and future engineering directions are prospected. This review aims to provide a reference for further engineering K. phaffii cell factory, which is supposed to facilitate its application in bioindustry.

A novel CRISPR/Cas9-hLacI donor adapting system for dsDNA-templated gene editing / 生物工程学报

During the gene editing process mediated by CRISPR/Cas9, precise genome editing and gene knock-in can be achieved by the homologous recombination of double-stranded DNA (dsDNA) donor template. However, the low-efficiency of homologous recombination in eukaryotic cells hampers the development and application of this gene editing strategy. Here, we developed a novel CRISPR/Cas9-hLacI donor adapting system (DAS) to enhance the dsDNA-templated gene editing, taking the advantage of the specific binding of the LacI repressor protein and the LacO operator sequence derived for the Escherichia coli lactose operon. The codon-humanized LacI gene was fused as an adaptor to the Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus lugdunensis Cas9 (SlugCas9-HF) genes, and the LacO operator sequence was used as the aptamer and linked to the dsDNA donor template by PCR. The Cas9 nuclease activity after the fusion and the homology-directed repair (HDR) efficiency of the LacO-linked dsDNA template were firstly examined using surrogate reporter assays with the corresponding reporter vectors. The CRISPR/Cas9-hLacI DASs mediated genome precise editing were further checked, and we achieved a high efficiency up to 30.5% of precise editing at the VEGFA locus in HEK293T cells by using the CRISPR/SlugCas9-hLacI DAS. In summary, we developed a novel CRISPR/Cas9-hLacI DAS for dsDNA-templated gene editing, which enriches the CRISPR/Cas9-derived gene editing techniques and provides a novel tool for animal molecular design breeding researches.

Application of single base editing technique in pig genetic improvement: a review / 生物工程学报

Traditional pig breeding has a long cycle and high cost, and there is an urgent need to use new technologies to revitalize the pig breeding industry. The recently emerged CRISPR/Cas9 genome editing technique shows great potential in pig genetic improvement, and has since become a research hotspot. Base editor is a new base editing technology developed based on the CRISPR/Cas9 system, which can achieve targeted mutation of a single base. CRISPR/Cas9 technology is easy to operate and simple to design, but it can lead to DNA double strand breaks, unstable gene structures, and random insertion and deletion of genes, which greatly restricts the application of this technique. Different from CRISPR/Cas9 technique, the single base editing technique does not produce double strand breaks. Therefore, it has higher accuracy and safety for genome editing, and is expected to advance the pig genetic breeding applications. This review summarized the working principle and shortcomings of CRISPR/Cas9 technique, the development and advantages of single base editing, the principles and application characteristics of different base editors and their applications in pig genetic improvement, with the aim to facilitate genome editing-assisted genetic breeding of pig.

Destruction of Being "Lifted": A Study on Klara and the Sun from the Perspective of Bioethics / 中国医学伦理学

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.

Legal Regulatory Approach of Germline Cell Gene Editing / 中国医学伦理学

Germline cell gene editing is a potentially revolutionary technology that can be used to treat and prevent many genetic diseases, such as cystic fibrosis and hemophilia. Although this technology has aroused widespread discussion in the medical community and the public, its legal regulatory approach has not yet been fully determined. By summarizing and studying the experience of relevant foreign laws and regulations, this paper analyzed and summarized the current legal situation in China, and explored three ethical topics of social equality, individual subjectivity, and intergenerational rights. Combining the existing legal norms and relevant ethical considerations, the legal regulatory approach for germline cell gene editing was proposed in view of the weak operability of the law, the blurred boundaries of the legislation, and the insufficiently perfect ethical review mechanism. To better ensure that the technology can benefit mankind on the premise of eliminating risks, the following suggestions were put forward: a specialized law should be formulated based on clear legislative principles to adjust and guide the improvement of the ethical review mechanism of germline cell gene editing. It is hoped that promoting the progress of germline cell gene editing technology through in-depth research, and providing useful reference for the formulation of relevant laws and ethical policies.

Progress in RNA-targeting and Gene Editing Therapies for Transthyretin Amyloidosis Cardiomyopathy / 罕见病研究

Transthyretin(TTR) protein is a tetramer protein, synthesized mainly by the liver. TTR can be misfolded and deposited as amyloid fibrilae and deposited in the myocardial interstroma leading to transthyroxin amyloidosis cardiomyopathy (ATTR-CM). ATTR-CM was included in China's First List of Rare Diseases. Therapeutic strategies for ATTR-CM include blocking TTR synthesis in the liver, stabilizing TTR tetramers and destroying TTR fibra. Small molecule drugs such as tafamidis and diflunisal offer new treatment options for patients. Chlorobenzolic acid became the first drug approved by the U.S. Food and Drug Administration for the treatment of ATTR-CM. Small interfering RNA(siRNA)patisiran and antisense oligonucleotide (ASO)inotersen block TTR expression in the liver and have been approved for the treatment of ATTR variant polyneuropathy (ATTRv-PN)and are in phase Ⅲ trials for the treatment of ATTR-CM. Other siRNA drugs, vutrisiran, and ASO, eplontersen, are being evaluated for clinical efficacy. This article reviews the development of RNA-targeted therapeutics and gene-editing drugs using CRISPR-Cas9.

Development strategy and clinical research progress of universal chimeric antigen receptor T-cell drugs / 中国药科大学学报

@#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.

Cloning and expression analysis of U6 promoters in Panax quinquefolius / 中国中药杂志

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.

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.

Application of live biotherapeutic products and perspective in the treatment of inherited metabolic disease / 生物工程学报

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.