CRISPR/Cas9 for overcoming drug resistance in solid tumors.
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ABSTRACT:
SUBMITTER: Saber A
PROVIDER: S-EPMC7214581 | biostudies-literature | 2020 Jun
REPOSITORIES: biostudies-literature
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Project description:Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9) screening is a simple screening method for locating loci under specific conditions, and it has been utilized in tumor drug resistance research for finding potential drug resistance-associated genes. This screening strategy has significant implications for further treatment of malignancies with acquired drug resistance. In recent years, studies involving genome-wide CRISPR/Cas9 screening have gradually increased. Here we review the recent application of genome-wide CRISPR/Cas9 screening for drug resistance, involving mitogen-activated protein kinase (MAPK) pathway inhibitors, poly (ADP-ribose) polymerase inhibitors (PARPi), alkylating agents, mitotic inhibitors, antimetabolites, immune checkpoint inhibitors (ICIs), and cyclin-dependent kinase inhibitors (CDKI). We summarize drug resistance pathways such as the KEAP1/Nrf2 pathway MAPK pathway, and NF-κB pathway. Also, we analyze the limitations and conditions for the application of genome-wide CRISPR/Cas9 screening techniques.
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Project description:Acute myeloid leukaemia (AML) continues to have a very high mortality and its treatment hasnot changed for more than 20 years.Here, we propose to apply genomic-wide screens to directly identify therapeutic vulnerabilitiesof AML using AML cells lines with pre-defined somatic mutations. Our studies will be basedon genome-wide CRISPR/CAs9 gRNA screens and will be used to identify drivers of drugresistance to standard chemotherapies.This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
2015-02-03 | E-ERAD-335 | biostudies-arrayexpress
Project description:The CRISPR-Cas9 system is attractive for gene therapy, as it allows for permanent genetic correction. However, as a new technology, Cas9 gene editing in clinical applications faces major challenges, such as safe delivery and gene targeting efficiency. Cas9 is a foreign protein to recipient cells; thus, its expression may prompt the immune system to eliminate gene-edited cells. To overcome these challenges, we have engineered a novel delivery system based on the helper-dependent adenoviral (HD-Ad) vector, which is capable of delivering genes to airway basal stem cells in vivo. Using this system, we demonstrate the successful co-delivery of both CRISPR-Cas9/single-guide RNA and the LacZ reporter or CFTR gene as donor DNA to cultured cells. HD-Ad vector genome integrity is compromised following donor DNA integration, and because the CRISPR-Cas9/single-guide RNA and donor DNA are carried on the same vector, CRISPR-Cas9 expression is concurrently eliminated. Thus, we show the feasibility of site-specific gene targeting with limited Cas9 expression. In addition, we achieved stable CFTR expression and functional correction in cultured cells following successful gene integration.
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Project description:Potato breeding can be redirected to a diploid inbred/F1 hybrid variety breeding strategy if self-compatibility can be introduced into diploid germplasm. However, the majority of diploid potato clones (Solanum spp.) possess gametophytic self-incompatibility that is primarily controlled by a single multiallelic locus called the S-locus which is composed of tightly linked genes, S-RNase (S-locus RNase) and multiple SLFs (S-locus F-box proteins), which are expressed in the style and pollen, respectively. Using S-RNase genes known to function in the Solanaceae gametophytic SI mechanism, we identified S-RNase alleles with flower-specific expression in two diploid self-incompatible potato lines using genome resequencing data. Consistent with the location of the S-locus in potato, we genetically mapped the S-RNase gene using a segregating population to a region of low recombination within the pericentromere of chromosome 1. To generate self-compatible diploid potato lines, a dual single-guide RNA (sgRNA) strategy was used to target conserved exonic regions of the S-RNase gene and generate targeted knockouts (KOs) using a Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (Cas9) approach. Self-compatibility was achieved in nine S-RNase KO T0 lines which contained bi-allelic and homozygous deletions/insertions in both genotypes, transmitting self compatibility to T1 progeny. This study demonstrates an efficient approach to achieve stable, consistent self-compatibility through S-RNase KO for use in diploid potato breeding approaches.
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Project description:L-asparaginase (ASNase) has been incorporated in the treatment of acute lymphoblastic leukemia for decades. However, intolerable toxicity due to increasing ASNase dosage has been observed in clinical trials with solid tumors. Here, we performed a genome-wide CRISPR-Cas9 functional screen in ASNase-resistant PC3 prostate cancer cells and identified SLC1A3, an aspartate transporter highly expressed in brain tissues but also in several tumor types. Interestingly, combined ASNase treatment and SLC1A3 inhibition caused cell cycle arrest and metabolic alterations in the urea cycle, nucleotide synthesis, energy production by both TCA cycle and glycolysis, and impaired lipid metabolism. Additionally, the introduction of SLC1A3 into SLC1A3-negative mouse and human breast cancer cells increased tumor and metastasis burden, and promoted ASNase tolerance in vitro and in vivo. These findings uncover an important role of SLC1A3 in ASNase resistance and indicated limited aspartate uptake for enhancement of ASNase efficacy with solid tumors.
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Project description:Multidrug resistance (MDR) has been extensively reported in colorectal cancer patients, which remains a major cause of chemotherapy failure. One of the critical mechanisms of MDR in colorectal cancer is the reduced intracellular drug level led by the upregulated expression of the ATP-binding cassette (ABC) transporters, particularly, ABCB1/P-gp. In this study, the CRISPR/Cas9 system was utilized to target ABCB1 in MDR colorectal cancer SW620/Ad300 cell line with ABCB1 overexpression. The results showed that stable knockout of ABCB1 gene by the CRISPR/Cas9 system was achieved in the MDR cancer cells. Reversal of MDR against ABCB1 chemotherapeutic drugs increased intracellular accumulation of [3H]-paclitaxel accumulation, and decreased drug efflux activity was observed in MDR SW620/Ad300 cells after ABCB1 gene knockout. Further tests using the 3D multicellular tumor spheroid model suggested that deficiency in ABCB1 restrained tumor spheroid growth and restore sensitivity to paclitaxel in MDR tumor spheroids. Overall, the CRISPR/Cas9 system targeting the ABCB1 gene can be an effective approach to overcome ABCB1-mediated MDR in colorectal cancer SW620/Ad300 cells.
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