Biology subjects

Yusa, K.

Publications and source records attributed to Yusa, K..

7 recordsLinked to original sources

bioRxiv2018

Tandem duplications lead to loss of fitness effects in CRISPR-Cas9 data

CRISPR-Cas9 gene-editing is widely used to study gene function and is being advanced for therapeutic applications. Structural rearrangements are a ubiquitous feature of cancers and their impact on CRISPR-Cas9 gene-editing has not yet been systematically assessed. Utilising CRISPR-Cas9 knockout screens for 163 cancer cell lines, we demonstrate that targeting tandem amplified regions is highly detrimental to cellular fitness, in contrast to amplifications caused by chromosomal duplications which have little to no effect. Genomically clustered Cas9 double-strand DNA breaks are associated with a strong gene-independent decrease in cell fitness. We systematically identified collateral vulnerabilities in 25% of cancer cells, introduced by tandem amplifications of tissue non-expressed genes. Our analysis demonstrates the importance of structural rearrangements in mediating the effect of CRISPR-Cas9-induced DNA damage, with implications for the use of CRISPR-Cas9 gene-editing technology, and how resulting collateral vulnerabilities are a generalisable strategy to target cancer cells.

genomics↗

bioRxiv2018

ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks

Mutations in the ATM tumor suppressor confer hypersensitivity to DNA-damaging agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase poison topotecan. Thus, we establish that loss of terminal components of the non-homologous end-joining (NHEJ) machinery or the BRCA1-A complex specifically confers topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase inhibitor olaparib is due to delayed homologous recombination repair at DNA-replication-fork-associated double-strand breaks (DSBs), resulting in toxic NHEJ-mediated chromosome fusions. Accordingly, restoring legitimate repair in ATM-deficient cells, either by preventing NHEJ DNA ligation or by enhancing DSB-resection by BRCA1-A complex inactivation, markedly suppresses this toxicity. Our work suggests opportunities for patient stratification in ATM-deficient cancers and when using ATM inhibitors in the clinic, and identifies additional therapeutic vulnerabilities that might be exploited when such cancers evolve drug resistance.\n\nOne Sentence SummaryATM counteracts toxic NHEJ at broken replication forks

cell biology↗

bioRxiv2018

JACKS: joint analysis of CRISPR/Cas9 knock-out screens

Genome-wide CRISPR/Cas9 knockout screens are revolutionizing mammalian functional genomics. Their range of applications remains limited by signal variability from different guide RNAs targeting the same gene, which confounds analysis, and dictates large experiment sizes. To address this problem, we report JACKS, a Bayesian method that jointly analyses screens performed with the same guide RNA library. Modeling the variable guide efficacies greatly improves hit identification, and allows a 2.5-fold reduction in required cell numbers without sacrificing performance compared to current analysis standards.

genomics↗

bioRxiv2017

Unsupervised correction of gene-independent cell responses to CRISPR-Cas9 targeting

Background: Genome editing by CRISPR-Cas9 technology allows large-scale screening of gene essentiality in cancer. A confounding factor when interpreting CRISPR-Cas9 screens is the high false-positive rate in detecting essential genes within copy number amplified regions of the genome. We have developed the computational tool CRISPRcleanR which is capable of identifying and correcting gene-independent responses to CRISPR-Cas9 targeting. CRISPRcleanR uses an unsupervised approach based on the segmentation of single-guide RNA fold change values across the genome, without making any assumption about the copy number status of the targeted genes.\n\nResults Applying our method to existing and newly generated genome-wide essentiality profiles from 15 cancer cell lines, we demonstrate that CRISPRcleanR reduces false positives when calling essential genes, correcting biases within and outside of amplified regions, while maintaining true positive rates. Established cancer dependencies and essentiality signals of amplified cancer driver genes are detectable post-correction. CRISPRcleanR reports sgRNA fold changes and normalised read counts, is therefore compatible with downstream analysis tools, and works with multiple sgRNA libraries.\n\nConclusions CRISPRcleanR is a versatile open-source tool for the analysis of CRISPR-Cas9 knockout screens to identify essential genes.

bioinformatics↗

bioRxiv2017

Genome-wide and high-density CRISPR-Cas9 screens identify point mutations in PARP1 causing PARP inhibitor resistance

PARP inhibitors (PARPi) target homologous recombination defective tumour cells via synthetic lethality. Genome-wide and high-density CRISPR-Cas9 \"tag, mutate and enrich\" mutagenesis screens identified single amino acid mutations in PARP1 that cause profound PARPi-resistance. These included PARP1 mutations outside of the DNA interacting regions of the protein, such as mutations in solvent exposed regions of the catalytic domain and clusters of mutations around points of contact between ZnF, WGR and HD domains. These mutations altered PARP1 trapping, as did a mutation found in a clinical case of PARPi resistance. These genetic studies reinforce the importance of trapped PARP1 as a key cytotoxic DNA lesion and suggest that interactions between non-DNA binding domains of PARP1 influence cytotoxicity. Finally, different mechanisms of PARPi resistance (BRCA1 reversion, PARP1, 53BP1, REV7 mutation) had differing effects on chemotherapy sensitivity, suggesting that the underlying mechanism of PARPi resistance likely influences the success of subsequent therapies.

cancer biology↗

bioRxiv2017

An unbiased reconstruction of the T helper cell type 2 differentiation network

T helper type 2 (Th2) cells are important regulators of our adaptive immune response, particularly the response against parasites, and have relevance for auto-immunity as well as tumour progression. This classic T helper type has been studied intensively, but not systematically. Using newly developed, genome-wide retroviral CRISPR knock-out (KO) technology, combined with RNA-seq, ATAC-seq and ChIP-seq, we have dissected the regulatory circuitry governing differentiation in these cells. During Th2 activation/differentiation approximately 4000 genes are perturbed, with at least 200 genes specifically associated with the Th2 program in mouse and human. We confirm previously known Th2 driver genes and have discovered several novel genes, including transcription factors, metabolic genes and potential receptors/cytokines, critical for Th2 function. Our study provides an atlas for, but not limited to, the Th2 regulatory network, pinpointing the key players of Th2 differentiation.

immunology↗

bioRxiv2017

A screen for combination therapies in BRAF/NRAS wild type melanoma identifies nilotinib plus MEK inhibitor as a synergistic combination

Despite recent therapeutic advances in the management of BRAFV600-mutant melanoma, there is still a compelling need for more effective treatments for patients who developed BRAF/NRAS wild type disease. Since the activity of single targeted agents is limited by innate and acquired resistance, we performed a high-throughput drug screen using 180 drug combinations to generate over 18,000 viability curves, with the aim of identifying agents that synergise to kill BRAF/NRAS wild type melanoma cells. From this screen we observed strong synergy between the tyrosine kinase inhibitor nilotinib and MEK inhibitors and validated this combination in an independent cell line collection. We found that AXL expression was associated with synergy to the nilotinib/MEK inhibitor combination, and that both drugs work in concert to suppress pERK. This finding was supported by genome-wide CRISPR screening which revealed that resistance mechanisms converge on regulators of the MAPK pathway. Finally, we validated the synergy of nilotinib/trametinib combination in vivo using patient-derived xenografts. Our results indicate that a nilotinib/MEK inhibitor combination may represent an effective therapy in BRAF/NRAS wild type melanoma patients.

cancer biology↗