Application prospects of organoids in organ transplantation

In recent years, organoid technology has become one of the major technological breakthroughs in biomedical field. As miniature organs constructed by three-dimensional culture of tissue stem cells in vitro, organoids are highly consistent with the source tissues in terms of tissue structures, cell types and functions, which serve as an ideal model for biomedical basic research, drug research and development and clinical precision medicine, and show important potential value in regenerative medicine. Organ transplantation is one of the most effective approaches to treat organ failure. However, the source of donor organs is currently limited, which could not meet the patients' needs. Identifying suitable graft substitutes is the key to breaking through the predicament. Organoids could be derived from the autologous tissues of patients. Multiple studies have demonstrated that organoids possess potent transplantation and repairing capabilities and may effectively avert the risk of immune rejection and tumorigenicity, etc. In this article, the development process and main application directions of organoid technology were summarized, and the application prospect and challenges of organoids in organ transplantation were reviewed and predicted.Copyright © 2022 Journal of Zhongshan University. All right reserved.

[Rapid detection and genotyping of SARS-CoV-2 Omicron BA.4/5 variants using a RT-PCR and CRISPR-Cas12a-based assay].

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.

Acceptance of genetic editing and of whole genome sequencing of human embryos by patients with infertility before and after the onset of the COVID-19 pandemic.

RESEARCH QUESTION: Has acceptance of heritable genome editing (HGE) and whole genome sequencing for preimplantation genetic testing (PGT-WGS) of human embryos changed after the onset of COVID-19 among infertility patients? DESIGN: A written survey conducted between April and June 2018 and July and December 2021 among patients at a university-affiliated infertility practice. The questionnaire ascertained the acceptance of HGE for specific therapeutic or genetic 'enhancement' indications and of PGT-WGS to prevent adult disease. RESULTS: In 2021 and 2018, 172 patients and 469 patients (response rates: 90% and 91%, respectively) completed the questionnaire. In 2021, significantly more participants reported a positive attitude towards HGE, for therapeutic and enhancement indications. In 2021 compared with 2018, respondents were more likely to use HGE to have healthy children with their own gametes (85% versus 77%), to reduce disease risk for adult-onset polygenic disorders (78% versus 67%), to increase life expectancy (55% versus 40%), intelligence (34% versus 26%) and creativity (33% versus 24%). Fifteen per cent of the 2021 group reported a more positive attitude towards HGE because of COVID-19 and less than 1% a more negative attitude. In contrast, support for PGT-WGS was similar in 2021 and 2018. CONCLUSIONS: A significantly increased acceptance of HGE was observed, but not of PGT-WGS, after the onset of COVID-19. Although the pandemic may have contributed to this change, the exact reasons remain unknown and warrant further investigation. Whether increased acceptability of HGE may indicate an increase in acceptability of emerging biomedical technologies in general needs further investigation.

Advances in CRISPR/Cas gene therapy for inborn errors of immunity.

Inborn errors of immunity (IEIs) are a group of inherited disorders caused by mutations in the protein-coding genes involved in innate and/or adaptive immunity. Hematopoietic stem cell transplantation (HSCT) is a mainstay definitive therapy for many severe IEIs. However, the lack of HLA-matched donors increases the risk of developing severe immunological complications. Gene therapy provides long-term clinical benefits and could be an attractive therapeutic strategy for IEIs. In this review, we describe the development and evolution of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins (Cas) gene-editing systems, including double-strand break (DSB)-based gene editing and DSB-free base editing or prime editing systems. Here, we discuss the advances in and issues associated with CRISPR/Cas gene editing tools and their potential as therapeutic alternatives for IEIs. We also highlight the progress of preclinical studies for the treatment of human genetic diseases, including IEIs, using CRISR/Cas and ongoing clinical trials based on this versatile technology.

Chemical Control of CRISPR Gene Editing via Conditional Diacylation Crosslinking of Guide RNAs.

Conditional control of RNA structure and function has emerged as an effective toolkit. Here, a strategy based on a one-step introduction of diacylation linkers and azide groups on the 2'-OH of RNA is advance. Selected from eight phosphine reagents, it is found that 2-(diphenylphosphino)ethylamine has excellent performance in reducing azides via a Staudinger reduction to obtain the original RNA. It is demonstrated that the enzymatic activities of Cas13 and Cas9 can be regulated by chemically modified guide RNAs, and further achieved ligand-induced gene editing in living cells by a controllable CRISPR/Cas9 system.

Progress in Respiratory Gene Therapy.

The prospect of gene therapy for inherited and acquired respiratory disease has energized the research community since the 1980s, with cystic fibrosis, as a monogenic disorder, driving early efforts to develop effective strategies. The fact that there are still no approved gene therapy products for the lung, despite many early phase clinical trials, illustrates the scale of the challenge: In the 1990s, first-generation non-viral and viral vector systems demonstrated proof-of-concept but low efficacy. Since then, there has been steady progress toward improved vectors with the capacity to overcome at least some of the formidable barriers presented by the lung. In addition, the inclusion of features such as codon optimization and promoters providing long-term expression have improved the expression characteristics of therapeutic transgenes. Early approaches were based on gene addition, where a new DNA copy of a gene is introduced to complement a genetic mutation: however, the advent of RNA-based products that can directly express a therapeutic protein or manipulate gene expression, together with the expanding range of tools for gene editing, has stimulated the development of alternative approaches. This review discusses the range of vector systems being evaluated for lung delivery; the variety of cargoes they deliver, including DNA, antisense oligonucleotides, messenger RNA (mRNA), small interfering RNA (siRNA), and peptide nucleic acids; and exemplifies progress in selected respiratory disease indications.

A Landscape of CRISPR/Cas Technique for Emerging Viral Disease Diagnostics and Therapeutics: Progress and Prospects.

Viral diseases have emerged as a serious threat to humanity and as a leading cause of morbidity worldwide. Many viral diagnostic methods and antiviral therapies have been developed over time, but we are still a long way from treating certain infections caused by viruses. Acquired immunodeficiency syndrome (AIDS) is one of the challenges where current medical science advancements fall short. As a result, new diagnostic and treatment options are desperately needed. The CRISPR/Cas9 system has recently been proposed as a potential therapeutic approach for viral disease treatment. CRISPR/Cas9 is a specialised, effective, and adaptive gene-editing technique that can be used to modify, delete, or correct specific DNA sequences. It has evolved into an advanced, configurable nuclease-based single or multiple gene-editing tool with a wide range of applications. It is widely preferred simply because its operational procedures are simple, inexpensive, and extremely efficient. Exploration of infectious virus genomes is required for a comprehensive study of infectious viruses. Herein, we have discussed the historical timeline-based advancement of CRISPR, CRISPR/Cas9 as a gene-editing technology, the structure of CRISPR, and CRISPR as a diagnostic tool for studying emerging viral infections. Additionally, utilizing CRISPR/Cas9 technology to fight viral infections in plants, CRISPR-based diagnostics of viruses, pros, and cons, and bioethical issues of CRISPR/Cas9-based genomic modification are discussed.

[CRISPR-based molecular diagnostics: a review].

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.

CRISPR-Cas13d effectively targets SARS-CoV-2 variants, including Delta and Omicron, and inhibits viral infection.

The recent pandemic of variants of concern (VOC) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the need for innovative anti-SARS-CoV-2 approaches in addition to vaccines and antiviral therapeutics. Here, we demonstrate that a CRISPR-Cas13-based strategy against SARS-CoV-2 can effectively degrade viral RNA. First, we conducted a cytological infection experiment, screened CRISPR-associated RNAs (crRNAs) targeting conserved regions of viruses, and used an in vitro system to validate functional crRNAs. Reprogrammed Cas13d effectors targeting NSP13, NSP14, and nucleocapsid transcripts achieved >99% silencing efficiency in human cells which are infected with coronavirus 2, including the emerging variants in the last 2 years, B.1, B.1.1.7 (Alpha), D614G B.1.351 (Beta), and B.1.617 (Delta). Furthermore, we conducted bioinformatics data analysis. We collected the sequence information of COVID-19 and its variants from China, and phylogenetic analysis revealed that these crRNA oligos could target almost 100% of the SARS-CoV family, including the emerging new variant, Omicron. The reprogrammed Cas13d exhibited high specificity, efficiency, and rapid deployment properties; therefore, it is promising for antiviral drug development. This system could possibly be used to protect against unexpected SARS-CoV-2 variants carrying multiple mutations.

An Insight into Modern Targeted Genome-Editing Technologies with a Special Focus on CRISPR/Cas9 and its Applications.

Genome-editing technology has enabled scientists to make changes in model organisms' DNA at the genomic level to get biotechnologically important products from them. Most commonly employed technologies for this purpose are transcription activator like effector nucleases (TALENs), homing-endonucleases or meganucleases, zinc finger nucleases (ZFNs), and clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9). Among these tools, CRISPR/Cas9 is most preferred because it's easy to use, has a small mutation rate, has great effectiveness, low cost of development, and decreased rate of advancement. CRISPR/Cas9 has a lot of applications in plants, animals, humans, and microbes. It also has applications in many fields such as horticulture, cancer, food biotechnology, and targeted human genome treatments. CRISPR technology has shown great potential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic to provide early and easy detection methods, possible treatment, and vaccine development. In the present review, genome-editing tools with their basic assembly and features have been discussed. Exceptional notice has been paid to CRISPR technology on basis of its structure and significant applications in humans, plants, animals, and microbes such as bacteria, viruses, and fungi. The review has also shed a little light on current CRISPR challenges and future perspectives.