Východiska: Editace genomu využívající CRISPR-Cas9 se v průběhu krátké doby zařadila mezi základní metody bio logického výzkumu. Tento nedávno objevený mechanizmus adaptivní antivirové obrany bakterií se podařilo přizpůsobit potřebám vědy a učinit z něj tak neocenitelný nástroj v manipulaci s DNA. K rozšíření metody přispěla především její jednoduchost a spolehlivost, s jakou ji lze využít. Pod pojmem editace genomu rozumíme úpravy genomové DNA cílené s přesností na jeden pár bází. V jednoduchosti zaměření enzymu na cílovou sekvenci se CRISPR-Cas9 zásadním způsobem liší od předchozích technologií. Na poli výzkumu nádorových onemocnění umožnil CRISPR-Cas9 vývoj řady modelových systémů vhodných pro studium karcinogeneze a testování léčiv. Z terapeutického hlediska našel CRISPR-Cas9 uplatnění v oblasti imunoterapie, a to zejména při ex vivo genetických modifikacích T lymfocytů pa cienta. Cíl: Terapeutický potenciál CRISPR-Cas9 v léčbě nádorových onemocnění se nyní snaží ověřit několik klinických studií. Na základě těchto studií jsme v článku shrnuli strategie použité při přípravě terapeutických nástrojů použitelných v protinádorové terapii. Závěr: Technologie CRISPR-Cas9 se ukazuje jako nepostradatelná v oblasti základního výzkumu při studiu funkce jednotlivých genů v procesu karcinogeneze. Využití metody v protinádorové terapii je více problematické. Před vlastní klinickou praxí je potřeba provést ještě řadu optimalizací týkajících se účinnosti, bezpečnosti a specifity CRISPR-Cas9.
Background: Genome editing using CRISPR-Cas9 has become one of the basic methods of biological research over a short period of time. This recently discovered system of adaptive immunity of bacteria has been adapted to the needs of science and has become a valuable tool for DNA manipulation. Its simplicity and reliability have contributed to widespread use of the method. Genome editing refers to targeted modifications of genomic DNA with single base pair accuracy. CRISPR-Cas9 differs significantly from previous technologies in the simplicity of directing the enzyme to the target sequence. In the field of cancer research, CRISPR-Cas9 has enabled the development of a number of models for the study of carcinogenesis and drug testing. From a therapeutic point of view, CRISPR-Cas9 has been applied in the field of immunotherapy, especially in ex vivo genetic modifications of the T-cells of patients. Aim: Currently, several clinical trials are trying to verify the therapeutic potential of CRISPR-Cas9. Based on these studies, we have summarised the strategies used in the preparation of therapeutic tools useful in cancer therapy. Conclusion: CRISPR-Cas9 appears to be crucial in basic research, particularly in the study of the function of individual genes involved in carcinogenesis. However, it will still be necessary to optimise the efficacy, safety and specificity of CRISPR-Cas9 before it is used in clinical practice.
- MeSH
- Immunotherapy methods MeSH
- Clinical Studies as Topic MeSH
- Humans MeSH
- Neoplasms * therapy MeSH
- CRISPR-Associated Protein 9 * immunology therapeutic use MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
CRISPR-Cas systems, which were originally developed by bacteria as a defence against viral infection, have recently significantly expanded the possibility of targeted interventions in the genome of many different organisms, including humans. The main fields, in which CRISPR-Cas systems are applied, are medicine, diagnostics, basic research, biotechnology and agriculture. However, their research is still in its infancy, as new systems are being discovered, known systems modified and the possibilities of their application expanding at unprecedent speed.
CRISPR/Cas je zkratka z anglického originálu Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated genes čili segmenty nahromaděných pravidelně rozmístěných krátkých palindromických repetic/CRISPRu asociovaných genů. Pomocí techniky CRISPR lze cíleně zasahovat do genetické informace vybraných organismů, umožňuje štěpení obou vláken dvojšroubovice DNA na konkrétním vybraném místě genomu. Rozvinutí použití umožnilo důkladné pochopení adaptivní imunity bakterií a archeí. Je známo několik variant této metody založených především na modifikacích nukleasy. Pomocí
CRISPR/Cas is a shortcut from English original: Clustered Regularly interspaced Short Palindromic Repeats / CRISPR‑associated genes. Using a CRISPR technique we can specifically manipulate with the hereditary information of selected organisms, the technique allows the cleavage of both strands of the double helix of DNA at a specific site in the genome. Developing application allows a deeper understanding of adaptive immunity of bacteria and archaea. Using the CRISPR method is possible to disable the function of specific genes, edit or regulate DNA transcription or mark selected location in the genome.
Genová editace je považována za největší pokrok v molekulárně genetických metodách od objevu polymerázové řetězové reakce. Díky této technologii dnes můžeme provádět cílené změny v nukleotidové sekvenci DNA s mnohem větší přesností a účinností, než bylo doposud možné. Využití nachází genová editace téměř ve všech biotechnologických oborech, neboť výrazně zjednodušuje studium funkce genů a biologických procesů. Nejpopulárnějším nástrojem genové editace je tzv. systém CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR Associated Protein 9), který si díky své relativně snadné konstrukci a vysoké účinnosti vydobyl výsadní postavení v molekulární biologii a jeho ohromný potenciál se již začíná uplatňovat i v translační medicíně. Cílem tohoto souhrnného článku je představení genové editace a shrnutí nejvýznamnějších aplikací této unikátní technologie ve výzkumu, diagnostice a léčbě hematologických onemocnění.
Genome editing is considered to be the biggest advance in molecular genetics since the discovery of the polymerase chain reaction. This method enables the introduction of changes in a target DNA sequence with unprecedented accuracy and efficiency. Since it greatly facilitates the study of genes and biological processes, it has been employed in basic research across all biotechnology fields. The CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR Associated Protein 9) system is the most widely used genome-editing platform. Due to its relatively easy construction and high efficiency, this system has revolutionized the field of molecular biology and also holds enormous potential in translational medicine. The aim of this review is to introduce genome-editing technique and summarize the most important applications of this unique technology in research, diagnostics and treatment of haematologic diseases.
- MeSH
- CRISPR-Cas Systems * MeSH
- Gene Editing methods MeSH
- Genetic Diseases, Inborn genetics therapy MeSH
- Hematologic Diseases * diagnosis genetics therapy MeSH
- Humans MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
BACKGROUND: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method). RESULTS: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. CONCLUSION: We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.
- MeSH
- Alleles * MeSH
- Blastocyst metabolism MeSH
- CRISPR-Cas Systems genetics MeSH
- Factor Analysis, Statistical MeSH
- Microinjections MeSH
- Mice, Knockout MeSH
- Methyl-CpG-Binding Protein 2 genetics metabolism MeSH
- CRISPR-Associated Protein 9 metabolism MeSH
- Regression Analysis MeSH
- Reproducibility of Results MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Multicenter Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
- MeSH
- Alleles MeSH
- CRISPR-Cas Systems * MeSH
- Gene Editing MeSH
- Mice MeSH
- CRISPR-Associated Protein 9 * metabolism MeSH
- Reproducibility of Results MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Letter MeSH
- Comment MeSH
- Publication type
- Meeting Abstract MeSH
Even though chemotherapy and immunotherapy emerged to limit continual and unregulated proliferation of cancer cells, currently available therapeutic agents are associated with high toxicity levels and low success rates. Additionally, ongoing multi-targeted therapies are limited only for few carcinogenesis pathways, due to continually emerging and evolving mutations of proto-oncogenes and tumor-suppressive genes. CRISPR/Cas9, as a specific gene-editing tool, is used to correct causative mutations with minimal toxicity, but is also employed as an adjuvant to immunotherapy to achieve a more robust immunological response. Some of the most critical limitations of the CRISPR/Cas9 technology include off-target mutations, resulting in nonspecific restrictions of DNA upstream of the Protospacer Adjacent Motifs (PAM), ethical agreements, and the lack of a scientific consensus aiming at risk evaluation. Currently, CRISPR/Cas9 is tested on animal models to enhance genome editing specificity and induce a stronger anti-tumor response. Moreover, ongoing clinical trials use the CRISPR/Cas9 system in immune cells to modify genomes in a target-specific manner. Recently, error-free in vitro systems have been engineered to overcome limitations of this gene-editing system. The aim of the article is to present the knowledge concerning the use of CRISPR Cas9 technique in targeting treatment-resistant cancers. Additionally, the use of CRISPR/Cas9 is aided as an emerging supplementation of immunotherapy, currently used in experimental oncology. Demonstrating further, applications and advances of the CRISPR/Cas9 technique are presented in animal models and human clinical trials. Concluding, an overview of the limitations of the gene-editing tool is proffered.
- MeSH
- CRISPR-Cas Systems * MeSH
- Gene Editing * MeSH
- Genetic Therapy * MeSH
- Immunotherapy, Adoptive MeSH
- Immunotherapy * MeSH
- Precision Medicine methods MeSH
- Clinical Trials as Topic MeSH
- Humans MeSH
- Disease Models, Animal MeSH
- Neoplasms etiology therapy MeSH
- Disease MeSH
- Drug Evaluation, Preclinical MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
