CRISPR screen
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V posledních letech došlo ke značnému pokroku v oblasti vývoje technik editace genomu a možnosti jejich klinického využití. Především objev adaptivního imunitního systému bakterií známého jako CRISPR a jeho rychlá implementace jako široce využitelné technologie způsobila zásadní převrat jak v základním, tak v aplikovaném biomedicínském výzkumu. Technologie CRISPR umožňuje editovat genom snadněji, rychleji a výrazně levněji než jakákoli jiná v současnosti dostupná technologie. Tímto se nabízí obrovský potenciál pro realizaci nových výzkumných přístupů a budoucí možnosti léčby nejrůznějších genetických onemocnění, vč. mnohočetného myelomu. Robustní využití CRISPR technologie v rámci genetických screeningů slibuje rychlejší identifikaci důležitých terapeutických cílů a současné odhalení biomarkerů s vysokou prediktivní hodnotou a doposud neznámých mechanizmů lékové rezistence. Výsledky takto směřovaného výzkumu tak mohou poskytnout nové diagnostické a prognostické přístupy, které umožňují přesnější stratifikaci pacientů pro personalizovanou léčbu s vyšší účinností. V tomto přehledném článku shrnujeme dosavadní znalosti technologie CRISPR s důrazem na její uplatnění při hledání nových terapeutických cílů, diagnostických markerů a genů zapojených do mechanizmů resistence na běžně používanou léčbu u mnohočetného myelomu. Závěrem prezentujeme potenciální budoucí využití technologie CRISPR v klinické praxi.
In the recent years, there was a remarkable advance in research and clinical implementation of the genome editing technologies. The most remarkable was a discovery of the bacterial adaptive immune system called CRISPR and its rapid transformation into a robust and broadly applicable technology that completely revolutionized both basic and applied biomedical research. Implementation of CRISPR makes genome modification easier, faster and significantly cheaper compare to any other currently available technology. It also offers a tremendous potential for desiging novel research approaches and future treatment options for various genetic diseases including multiple myeloma. The hightroughput use of CRISPR in pooled screen formats promises faster identification and validation of valuable drug targets together with revealing high-confidence biomarkers and unknown resistance mechanisms. This can provide clinicians with new diagnostic and prognostic tolls and ultimately allow more accurate patient stratification for personalised treatment with better eficacy. In this review, we summarize current knowledge about the CRISPR technology and focus especially on its impact in exploring gene functions, screening for novel drug targets, diagnostic markers and genes involved in resistance to commonly used drug in the treatment of multiple myeloma. Finally, we also highlight a potential future use of CRISPR in actual clinical practise.
CFTR is a membrane protein that functions as an ion channel. Mutations that disrupt its biosynthesis, trafficking or function cause cystic fibrosis (CF). Here, we present a novel in vitro model system prepared using CRISPR/Cas9 genome editing with endogenously expressed WT-CFTR tagged with a HiBiT peptide. To enable the detection of CFTR in the plasma membrane of live cells, we inserted the HiBiT tag in the fourth extracellular loop of WT-CFTR. The 11-amino acid HiBiT tag binds with high affinity to a large inactive subunit (LgBiT), generating a reporter luciferase with bright luminescence. Nine homozygous clones with the HiBiT knock-in were identified from the 182 screened clones; two were genetically and functionally validated. In summary, this work describes the preparation and validation of a novel reporter cell line with the potential to be used as an ultimate building block for developing unique cellular CF models by CRISPR-mediated insertion of CF-causing mutations.
- MeSH
- buněčná membrána metabolismus MeSH
- buněčné linie MeSH
- CRISPR-Cas systémy genetika MeSH
- cystická fibróza * genetika metabolismus MeSH
- lidé MeSH
- protein CFTR * genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články 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 systémy * MeSH
- editace genu * MeSH
- genetická terapie * MeSH
- imunoterapie adoptivní MeSH
- imunoterapie * MeSH
- individualizovaná medicína metody MeSH
- klinické zkoušky jako téma MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- nádory etiologie terapie MeSH
- nemoc MeSH
- preklinické hodnocení léčiv MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
CRISPR-Cas gene editing holds substantial promise in many biomedical disciplines and basic research. Due to the important functional implications of non-histone chromosomal protein HMG-14 (HMGN1) in regulating chromatin structure and tumor immunity, gene knockout of HMGN1 is performed by CRISPR in cancer cells and the following proteomic regulation events are studied. In particular, DIA mass spectrometry (DIA-MS) is utilized, and more than 6200 proteins (protein- FDR 1%) and more than 82 000 peptide precursors are reproducibly measured in the single MS shots of 2 h. HMGN1 protein deletion is confidently verified by DIA-MS in all of the clone- and dish- replicates following CRISPR. Statistical analysis reveals 147 proteins change their expressions significantly after HMGN1 knockout. Functional annotation and enrichment analysis indicate the deletion of HMGN1 induces histone inactivation, various stress pathways, remodeling of extracellular proteomes, cell proliferation, as well as immune regulation processes such as complement and coagulation cascade and interferon alpha/ gamma response in cancer cells. These results shed new lights on the cellular functions of HMGN1. It is suggested that DIA-MS can be reliably used as a rapid, robust, and cost-effective proteomic-screening tool to assess the outcome of the CRISPR experiments.
- MeSH
- chromatin fyziologie MeSH
- CRISPR-Cas systémy MeSH
- delece genu * MeSH
- editace genu metody MeSH
- HeLa buňky MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- proliferace buněk genetika MeSH
- protein HMGN1 genetika MeSH
- proteomika metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Purines are essential molecules for nucleic acid synthesis and are the most common carriers of chemical energy in all living organisms. The cellular pool of purines is maintained by the balance between their de novo synthesis (DNPS), recycling and degradation. DNPS includes ten reactions catalysed by six enzymes. To date, two genetically determined disorders of DNPS enzymes have been described, and the existence of other defects manifested by neurological symptoms and the accumulation of DNPS intermediates in bodily fluids is highly presumable. In the current study, we prepared specific recombinant DNPS enzymes and used them for the biochemical preparation of their commercially unavailable substrates. These compounds were used as standards for the development and validation of quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). To simulate manifestations of known and putative defects of DNPS we prepared CRISPR-Cas9 genome-edited HeLa cells deficient for the individual steps of DNPS (CR-cells), assessed the substrates accumulation in cell lysates and growth media and tested how the mutations affect assembly of the purinosome, the multi-enzyme complex of DNPS enzymes. In all model cell lines with the exception of one, an accumulation of the substrate(s) for the knocked out enzyme was identified. The ability to form the purinosome was reduced. We conclude that LC-MS/MS analysis of the dephosphorylated substrates of DNPS enzymes in bodily fluids is applicable in the selective screening of the known and putative DNPS disorders. This approach should be considered in affected individuals with neurological and neuromuscular manifestations of unknown aetiology. Prepared in vitro human model systems can serve in various studies that aim to provide a better characterization and understanding of physiology and pathology of DNPS, to study the role of each DNPS protein in the purinosome formation and represent an interesting way for the screening of potential therapeutic agents.
- MeSH
- chromatografie kapalinová MeSH
- CRISPR-Cas systémy * MeSH
- HeLa buňky MeSH
- lidé MeSH
- multienzymové komplexy chemie genetika metabolismus MeSH
- mutace MeSH
- puriny biosyntéza metabolismus MeSH
- substrátová specifita MeSH
- tandemová hmotnostní spektrometrie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The CRISPR/Cas9 technique is widely used in experimentation with human cell lines as well as with other model systems, such as mice Mus musculus, zebrafish Danio reiro, and the fruit fly Drosophila melanogaster. However, publications describing the use of CRISPR/Cas9 for genome editing in non-model organisms, including non-model insects, are scarce. The introduction of this relatively new method presents many problems even for experienced researchers, especially with the lack of procedures to tackle issues concerning the efficiency of mutant generation. Here we present a protocol for efficient genome editing in the non-model insect species Pyrrhocoris apterus. We collected data from several independent trials that targeted several genes using the CRISPR/Cas9 system and determined that several crucial optimization steps led to a remarkably increased efficiency of mutant production. The main steps are as follows: the timing of embryo injection, the use of the heteroduplex mobility assay as a screening method, in vivo testing of sgRNA efficiency, and G0 germline mosaicism screening. The timing and the method of egg injections used here need to be optimized for other species, but other here-described optimization solutions can be applied immediately for genome editing in other insect species.
- Publikační typ
- časopisecké články MeSH
B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.
- MeSH
- B-buněčný lymfom genetika patologie MeSH
- buněčné klony MeSH
- CRISPR-Cas systémy genetika MeSH
- genetické asociační studie MeSH
- genetické testování metody MeSH
- genová dávka MeSH
- geny nádorové MeSH
- lidé MeSH
- myši inbrední C57BL MeSH
- myši transgenní MeSH
- receptory antigenů B-buněk metabolismus MeSH
- regulace genové exprese u nádorů MeSH
- reprodukovatelnost výsledků MeSH
- transpozibilní elementy DNA genetika MeSH
- tumor supresorové geny MeSH
- ztráta heterozygozity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Maintenance of genome integrity via repair of DNA damage is a key biological process required to suppress diseases, including Fanconi anemia (FA). We generated loss-of-function human haploid cells for FA complementation group C (FANCC), a gene encoding a component of the FA core complex, and used genome-wide CRISPR libraries as well as insertional mutagenesis to identify synthetic viable (genetic suppressor) interactions for FA. Here we show that loss of the BLM helicase complex suppresses FANCC phenotypes and we confirm this interaction in cells deficient for FA complementation group I and D2 (FANCI and FANCD2) that function as part of the FA I-D2 complex, indicating that this interaction is not limited to the FA core complex, hence demonstrating that systematic genome-wide screening approaches can be used to reveal genetic viable interactions for DNA repair defects.
- MeSH
- buněčné linie MeSH
- CRISPR-Cas systémy MeSH
- DNA-helikasy genetika MeSH
- Fanconiho anemie genetika MeSH
- haploidie MeSH
- HEK293 buňky MeSH
- helikasy RecQ genetika MeSH
- inzerční mutageneze MeSH
- lidé MeSH
- NAD(P)H dehydrogenasa (chinon) genetika MeSH
- oprava DNA genetika MeSH
- poškození DNA MeSH
- protein FANCC genetika MeSH
- protein FANCD2 genetika MeSH
- proteiny FANC genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- MeSH
- antivirové látky farmakologie MeSH
- biopaliva MeSH
- biotechnologie metody MeSH
- CRISPR-Cas systémy MeSH
- editace genu metody MeSH
- kongresy jako téma MeSH
- lidé MeSH
- manipulace s potravinami metody MeSH
- potravinářská mikrobiologie MeSH
- preklinické hodnocení léčiv metody MeSH
- střevní mikroflóra MeSH
- syntetická biologie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- úvodní články MeSH
- úvodníky MeSH
