Ginsenoside Rd inhibits migration and invasion of tongue cancer cells through H19/miR-675-5p/CDH1 axis

Abstract Objective Tongue squamous cell carcinoma (TSCC) is an oral cancer, with high malignancy and frequent early migration and invasion. Only a few drugs can treat tongue cancer. Ginsenoside Rd is a ginseng extract with anti-cancer effects. Many noncoding RNAs are abnormally expressed in tongue cancer, thus influencing its occurrence and development. H19 and miR-675-5p can promote cancer cell growth. This study aimed to analyze the regulation effect of ginsenoside Rd on H19 and miR-675-5p in tongue cancer. Methodology We used CCK8 and flow cytometry to study the growth and apoptosis. Transwell assay was used to assess invasion; wound-healing assay to assess migration; and colony formation assays to test the ability of cells to form colonies. H19, miR-675-5p, and CDH1 expressions were analyzed by qPCR. E-cadherin expression was detected using western blot. CRISPR/cas9 system was used for CDH1 knockout. Results Ginsenoside Rd inhibited the growth and increased the apoptosis of SCC9 cells. Ginsenoside Rd also inhibited the migration and invasion of SCC9 cells. H19 and miR-675-5p were highly expressed, while CDH1 and E-cadherin expressions were low. H19 and miR-675-5p promoted SCC9 metastasis. In contrast, CDH1 and E-cadherin inhibited the metastasis of SCC9 cells. Bioinformatics analysis showed that miR-675-5p was associated with CDH1. H19 and miR-675-5p expressions decreased after ginsenoside Rd treatment, while CDH1 and E-cadherin expressions increased. Conclusions Ginsenoside Rd inhibits tongue cancer cell migration and invasion via the H19/miR-675-5p/CDH1 axis.

Recent advances in CRISPR-related transposable elements / 生物工程学报

A new wave of research has been inspired by the CRISPR-Cas system with respect to their application in genome editing. The CRISPR-Cas system can not only be applied in gene knockout and insertion, but also be used in base editing, transcriptional regulation and recombination of gene clusters. However, the low efficiency of homology-directed repair (HDR) limits its application. Unlike the CRISPR-Cas system, mobile genetic elements (MGE) can insert DNA fragments into cell chromosomes without the aid of HDR. Recently, it is reported that CRISPR-related transposable elements can guide targeted DNA insertion. Their transposition mechanisms and reprogramming abilities have brought novel opportunities to the development of this field. This review summarized the research progress and application development of natural CRISPR-related transposable elements in recent years, as well as the applications of fused dCas9-transposase. It proposed the application prospects and potential challenges of CRISPR-related transposable elements in the future, which provided a reference for the development direction of gene editing tools.

Application of CRISPR/Cas9 gene editing technology in the construction of animal models of ophthalmic diseases / 中华实验眼科杂志

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) technology is a gene editing technology that uses RNA to guide endonucleases.This technology is rapidly used in gene editing and disease gene therapy in multiple species because of its easy operation, precise targeting, short cycle, and high gene knockout efficiency.At present, the corneal dystrophy model ( UBIAD1, TGF- β R124C gene mutations), glaucoma model ( MYOC Y435H, OPTN E50K and PMEL gene mutations), cataract model ( GJA8, KPNA4, C- MAF, AQP5 and PIKFYVE gene mutations), Leber congenital amaurosis animal model ( KCNJ13 and LCA5 gene mutations), retinblastoma animal model ( RB1/ RBL gene mutations) and retinitis pigmentosa models ( HKDC1, C8ORF37, CERKL, PRCD, ASRGL1, LRAT and PDE6B gene mutations) have been constructed by using this technology.The role of MFRP, CPAMD8, Pax6, and FREM genes in animal eye development has been further confirmed via this technology.The application of CRISPR/Cas9 gene editing technology in the construction of animal models of ophthalmic diseases was reviewed in this article.

Principios y aplicaciones médicas de la edición de genes mediante CRISPR/Cas

RESUMEN La tecnología CRISPR/Cas es un método simple, rápido y extremadamente eficiente para la edición de genes. Con el objetivo de describir los principios y aplicación médica de dicha tecnología, se realizó una revisión bibliográfica en Pubmed, SciELO, Google académico y Cochrane Library, con los descriptores "edición de genes", "edición del genoma", "sistemas CRISPR-Cas" y "proteína 9 asociada a CRISPR". El sistema CRISPR/Cas9 comprende una endonucleasa Cas y dos tipos de RNA. Cas corta el DNA del fago invasor en segmentos, los cuales se integran a la secuencia CRISPR como espaciadores. Posteriormente, la secuencia CRISPR se transcribe para generar crRNA y tracrRNA, que forman una estructura de RNA de doble hélice que recluta Cas para el clivaje. La introducción del sistema al interior celular se produce con plásmidos, RNA o ribonucleoproteínas. Una secuencia de localización nuclear permite que CRISPR/Cas9 entre al núcleo. La tecnología CRISPR/Cas9 es una eficiente herramienta para la edición precisa de genes con gran impacto en la investigación científica.

ABSTRACT CRISPR/Cas technology is a simple, fast and extremely efficient method for gene editing. In order to describe the principles and medical application of this technology, a bibliographic review was carried out in Pubmed, SciELO, academic Google and the Cochrane Library, with the descriptors "gene editing", "genome editing", "CRISPR-Cas systems", and "CRISPR-associated protein 9". The CRISPR/Cas9 system comprises a Cas endonuclease and two types of RNA. Cas cuts the invading phage DNA into segments, which are integrated into the CRISPR sequence as spacers. Subsequently, the CRISPR sequence is transcribed to generate crRNA and tracrRNA, which form a double-stranded RNA structure that recruits Cas for cleavage. The introduction of the system into the cell interior occurs with plasmids, RNA or ribonucleoproteins. A nuclear localization sequence allows CRISPR / Cas9 to enter the nucleus. CRISPR/Cas9 technology is an efficient tool for precise gene editing with great impact on scientific research.

Implicações da obsolescência dos seres humanos modificados geneticamente em virtude dos avanços tecnológicos da edição genética / Implications of the obsolescence of genetically modified human beings due to technological advances in the field of genetic editing / Implicaciones de la obsolescencia de los seres humanos genéticamente modificados debido a los avances tecnológicos en el campo de la edición genética

Objetivo: analisar, sob uma perspectiva bioética, como a utilização da edição genética, uma vez incorporada à prática médica, poderá causar obsolescência genética aos seres humanos modificados e quais seriam as implicações para o indivíduo obsoleto, bem como os reflexos dessa problemática no direito à saúde. Metodologia:foi aplicado o método hipotético-dedutivo com abordagem qualitativa, tendo como fundamento trabalhos científicos das áreas de genética e bioética publicados em periódicos nacionais e internacionais. Resultado: o surgimento de nova tecnologia de edição genética denominada sistema CRISPR-Cas representa grande avanço científico, uma vez que se trata de instrumento mais acessível e eficiente na edição genética. As modificações genéticas em seres humanos voltadas não apenas para a terapêutica, mas também para o melhoramento do próprio genoma, tornam-se possíveis e, com o surgimento de pessoas com aperfeiçoamento genético no futuro, vem à tona o dilema ético da obsolescência desses indivíduos, bem como possíveis implicações na seara jurídica advindas de melhoramentos genéticos. Conclusão:a edição genética voltada para o aperfeiçoamento poderá causar obsolescência dos seres humanos em determinadas searas, incluindo o aprofundamento de iniquidades em questões de saúde. Essa obsolescência guarda aproximação com a ideia habermasiana da indistinção entre pessoas e produtos e pode causar profunda angústia existencial àqueles indivíduos modificados geneticamente quando essas modificações se tornarem ultrapassadas.

Objective: to analyze, from a bioethical perspective, how the use of genetic editing, once incorporated into medical practice, may cause genetic obsolescence to modified human beings and what would be the implications for the obsolete individual, as well as the reflections of this problem to the right to healthcare.Methodology:hypothetical-deductive method, based on scientific works in the areas of genetics and bioethicspublished in national and international journals. Result:the emergence of new genetic editing technology called the CRISPR-Cas system represents a major scientific advance, since it is a more accessible and efficient instrument in genetic editing. Genetic modifications in human beings focus not only therapeutics, but also on improving the genome itself. With the emergence of people with genetic improvement in the future, the ethical dilemma of the obsolescence of these individuals comes to the fore, as well as possible implications in the legal field arising from genetic improvements. Conclusion:genetic edition aimed at improvement may cause human obsolescence in certain fields, including the deepening of inequities in health issues. This obsolescence is close to the Habermasian idea of indistinction between people and products and can cause deep existential anguish to those genetically modified individuals when these changes become outdated.

Objetivo: analizar, desde una perspectiva bioética, cómo el uso de la edición genética, una vez incorporada a la práctica médica, puede ocasionar la obsolescencia genética de seres humanos modificados y cuáles serían las implicaciones para el individuo obsoleto, así como las reflexiones de esta problemática sobre el derecho a la salud.Metodología: se aplicó el método hipotético-deductivo con enfoque cualitativo, basado en trabajos científicos en las áreas de genética y bioética publicados en revistas nacionales e internacionales.Resultado: la aparición de una nueva tecnología de edición genética denominada sistema CRISPR-Cas representa un gran avance científico, ya que es un instrumento más accesible y eficiente en la edición genética. Las modificaciones genéticas en los seres humanos centradas no solo en la terapia, sino también en la mejora del genoma en sí, se hacen posibles y, con la aparición de personas con mejora genética en el futuro, el dilema ético de la obsolescencia de estos individuos pasa a primer plano. así como las posibles implicaciones en el ámbito jurídico derivadas de las mejoras genéticas.Conclusión:la edición genética orientada a la mejora puede provocar la obsolescencia humana en determinados campos, incluida la profundización de las inequidades en materia de salud. Esta obsolescencia se acerca a la idea habermasiana de indistinción entre personas y productos y puede causar una profunda angustia existencial en aquellos individuos genéticamente modificados cuando estos cambios se vuelven obsoletos.

Edição de humanos por meio da técnica do Crispr-cas9: entusiasmo científico e inquietações éticas / Human editing using the Crispr-cas9 technique: scientific enthusiasm and ethical concerns

RESUMO Esta reflexão crítica se dá em torno da ética científica do Crispr-Cas9, e estrutura-se em cinco momentos: 1) inicia-se recordando o filme 'Gattaca' e o experimento de edição de embriões humanos por meio da técnica Crispr-Cas9, protagonizado por He Jiankui, no final de 2018, na China, e que chocou o mundo científico; 2) a seguir, faz-se uma incursão em busca de compreensão sobre a descoberta do Crispr-Cas9 e que tipo de implicações éticas traz à humanidade; 3) nessa etapa, fazem-se considerações em torno da pesquisa científica dessa revolucionária técnica de edição de genes, que tem que continuar e evoluir, mas respeitando diretrizes éticas; 4) necessita-se de diretrizes éticas para a governança das técnicas de edição do genoma humano - alguns princípios éticos gerais são propostos; 5) finaliza-se a reflexão apontando para a necessidade de fazer uma ciência com consciência, prudência e responsabilidade, para construir um futuro com esperança ética, evitando o alarmismo apocalíptico (tecnofobia) de um lado, bem como o utopismo tecnológico ingênuo (tecnoutopia) de outro.

ABSTRACT This critical reflection takes place around the scientific ethics of Crispr-Cas9, and is structured in five moments: 1) it starts by remembering the film 'Gattaca' and the experiment of editing human embryos using the Crispr-Cas9 technique, by He Jiankui, in late 2018, in China, and which shocked the scientific world; 2) next, there is an inroad in search of understanding about the discovery of Crispr-Cas9 and what kind of ethical implications it brings to humanity; 3) at this stage, considerations are made around the scientific research of this revolutionary gene editing technique, which has to continue and evolve, while respecting ethical guidelines; 4) ethical guidelines are needed for the governance of human genome editing techniques - some general ethical principles are proposed; 5) the reflection ends by pointing to the need to make science with conscience, prudence and responsibility, to build a future with ethical hope, avoiding apocalyptic alarmism (technophobia) on the one hand, as well as naive technological utopianism (technoutopia) from another.

Research progress of diagnosis platforms based on CRISPR/Cas system / 中华检验医学杂志

At present, nucleic acid testing technology has been widely used in clinical laboratory diagnosis. Conventional detection technique such as real-time PCR is complicated, time consuming, and dependent on specific instruments. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system is an adaptive immune defense system against viruses in bacteria and archaea, which has been developed into a powerful technology for genome editing. Recently, the leading groups engaged in CRISPR have set up new tools for nucleic acid detection based on Cas13a, Cas12a and newly discovered protein-Cas14, which plays an important role in rapid diagnosis of infectious diseases, detection of gene mutations in cancer and genotyping. Since they are ultrasensitive, specific, rapid and cost-effective, it is expected to bring great potential for molecular diagnosis. In this review, the mechanism of CRISPR/Cas system and the principle, the applications of the newly-developed diagnostic platforms are introduced. What′s more, the advantages, limitations and prospects of the technologies are summarized.

The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells

The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting the EGFP gene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells.

The technical development and application prospects of CRISPR/Cas9 in laboratory medicine / 中华检验医学杂志

Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9),a cluster of regularly spaced short palindromic repeats,is a natural immune defense system for bacteria and archaea to identify themselves and exogenous invading DNA fragments,protecting them from viruses.In recent years,CRISPR/Cas9 has become a revolutionary gene editing tool.Its specific targeted spot-cutting ability also plays an important role in nucleic acid detection,bacterial typing,etc.,and has shown great application potential in the field of medical testing.Based on the latest researches,this paper reviews the progress of CRISPR/Cas9 application in the new techniques of nucleic acid detection,pathogen typing and bacterial evolution in laboratory medicine,and also summarizes the application prospect of CRISPR technology in the field of laboratory medicine.

Genetics of Alzheimer's Disease

Alzheimer's disease (AD) related genes have been elucidated by advanced genetic techniques. Familial autosomal dominant AD genes founded by linkage analyses are APP, PSEN1, PSEN2, ABCA7, and SORL1. Genome-wide association studies have found risk genes such as ABCA7, BIN1, CASS4, CD33, CD2AP, CELF1, CLU, CR1, DSG2, EPHA1, FERMT2, HLA-DRB5-HLA-DRB1, INPP5D, MEF2C, MS4A6A/MS4A4E, NME8, PICALM, PTK2B, SLC24A4, SORL1, and ZCWPW1. ABCA7, SORL1, TREM2, and APOE are proved to have high odds ratio (>2) in risk of AD using next generation sequencing studies. Thanks to the promising genetic techniques such as CRISPR-CAS9 and single-cell RNA sequencing opened a new era in genetics. CRISPR-CAS9 can directly link genetic knowledge to future treatment. Single-cell RNA sequencing are providing useful information on cell biology and pathogenesis of diverse diseases.

The establishment of zebrafish Dio3b gene knockout model / 中华内分泌代谢杂志

Objective To set up the model of deiodinase ( Dio) 3b-/- zebrafish and to observe the effect of which on embryo development. Methods The zebrafish model of Dio3b-/- was set up by CRISPR-Cas9 gene editing technology, PCR and sequencing was used to confirm the efficiency of deletion. The heart rate of embryos at 48 hours post fertilization was counted. The locomotor activity of 5-7 days post fertilization larve was detected using behavior tracking system. Results The model of Dio3b knockout zebrafish was set up successfully. The heart rate of embryos Dio3b-/- increased ( P＜0. 001) and the locomotor activity of 5-7 days post fertilization larves lacking Dio3b gene increased (P＜0.05) significantly compared with that of wild type control respectively. Conclusion The deletion of zebrafish Dio3b gene results in the phenotype of hyperthyroidism and the model of Dio3b-/- is proper for studying the effect of partial excess thyroid hormone on embryo development.

Applications of CRISPR/Cas9 genome editing technology in gene therapy for hereditary eye diseases / 中华实验眼科杂志

Clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR associated nuclease (Cas) system is an adaptive immune system that confers resistance to exogenous virus or plasmid in bacteria and archaea,over the 30 years since its discovery,researchers have a better understanding of its immune processes in vivo and the mechanisms of gene editing by using its function. Researches found that CRISPR/Cas9 system modified from typeⅡCRISPR/Cas may edit genome accurately and effectively. In recent years,with the progress and development of gene sequencing technology,it is more explicit to make genetic diagnosis of a variety of hereditary eye diseases,and with the improvement of specificity for Cas9 in eukaryotic cells,gene editing is showing a great potential in the field of treating hereditary eye diseases. At present,the application of CRISPR/Cas9 gene editing technology has extended to the gene therapy of some hereditary eye diseases, such as congenital cataract, congenital glaucoma, retinitis pigmentosa (RP),congenital corneal dystrophy,Leber congenital amaurosis (LCA) and Usher syndrome. Besides,the combination of CRISPR/Cas9 gene editing technology with adeno-associated virus vectors (AAV) and induced pluripotent stem cells (iPSCs) research offers more possibilities and new approaches for the treatment of hereditary diseases. This article reviewed the mechanism of CRISPR/Cas9 and its applications in gene therapy for hereditary eye diseases.

Intervenções genéticas em seres humanos: aspectos éticos e jurídicos / Genetic interventions in humans: ethical and legal aspects / Intervenciones genéticas en seres humanos: aspectos éticos y jurídicos

Os avanços da biotecnologia podem gerar crescentes e significativas transformações na vida e na saúde de seres humanos. Em que pese os relevantes benefícios que advêm do uso das respectivas técnicas, os riscos inerentes e o desconhecimento de limites são motivos de inquietações e divergências, especialmente no que diz respeito às intervenções genéticas. Exemplo recente é o uso do sistema CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats – Cas9 associated) para introduzir mutações específicas no genoma humano. O presente artigo tem como escopo abranger aspectos éticos e jurídicos que permeiam a medicina genética corretiva. Para desenvolver o objetivo proposto, utilizamos o método qualitativo de abordagem, mediante pesquisa documental sobre o assunto. Como resultado, apuramos a importância da (des)construção de parâmetros para nortear o desenvolvimento e a implementação das técnicas de intervenção na genética humana, de modo a promover maior segurança à pesquisa e à humanidade.(AU)

Advances in biotechnology may generate increasing and significant changes in human life and health. Despite the significant benefits wich come from the use of technics, the inherent risks and the lack of knowledge of limits are causes for concerns and disagreements, especially with regard to genetic interventions. A recent example is the use of CRISPR-Cas9(Clustered Regularly Interspaced Short Palindromic Repeats, associated with protein 9) system to introduce specific mutations in human genome. This article aims to inquire into ethical and legal aspects that permeate the corrective genetic medicine. In order to develop theproposed objective, the qualitative method of approach is used with documentary research on the subject. As result, it was found the importance of (de)construction of parameters to guide the development and implementation of techniques of intervention in human genetics, so thatgreater security is promoted to research and to mankind.(AU)

Los avances d la biotecnología pueden generar crecientes y significativas transformaciones en la vida y en la salud de seres humanos. En que pese a los relevantes beneficios que advienendel uso de las respectivas técnicas, los riesgos inherentes y el desconocimiento de límites son motivos de inquietudes y divergencias, especialmente en lo que se refiere a las intervenciones genéticas. Ejemplo reciente es el uso del sistema CRISPR-Cas9 (Clustered RegularlyInterspaced Short Palindromic Repeats – Cas9 associated) para introducir mutaciones específicas en el genoma humano. El presente artículo tiene como ámbito abarcar aspectos éticos y jurídicos que permean la medicina genética correctiva. Para desarrollar el objetivopropuesto, utilizamos el método cualitativo de abordaje, mediante investigación documental sobre el asunto. Como resultado, verificamos la importancia de la (des)construcción de parámetros para guiar el desarrollar y la implementación de las técnicas de intervención en la genética humana, de modo a promover más seguridad a la investigación y a la humanidad.(AU)