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ABSTRACT: Candidate transmission survival genome of Mycobacterium tuberculosis-CRISPRi Screen II

PROVIDER: PRJNA1208562 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR31945403_1.fastq.gz	Fastqsanger.gz
	SRR31945403_2.fastq.gz	Fastqsanger.gz
	SRR31945404_1.fastq.gz	Fastqsanger.gz
	SRR31945404_2.fastq.gz	Fastqsanger.gz
	SRR31945405_1.fastq.gz	Fastqsanger.gz

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Project description:Investigation of whole genome gene expression level changes in Mycobacterium tuberculosis treated with the DHFR inhibitor WR99210, compared to untreated cells. The antimycobacterial properties of WR99210 are further described in Gerum, A., Ulmer, J., Jacobus, D., Jensen, N., Sherman, D., and C. Sibley. 2002. Novel Saccharomyces cerevisiae screen identifies WR99210 analogues that inhibit Mycobacterium tuberculosis dihydrofolate reductase. Antimicrob Agents Chemother 46(11):3362-3369 [PMID:12384337] A nine chip experiment in which total RNA was collected from H37Rv after treatment with 100 uM WR99210 for 0, 4, and 24 hours. Three separate cultures/biological replicates were interrogated for both treated and untreated cultures. Labeled cDNA was hybridized to NimbleGen spotted oligo tiling arrays covering both strands of the M. tuberculosis genome at ~200 bp intervals between 60-mer probes.

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CRISPRi Screen of Long Non-coding RNAs Identifies LINC03045 Regulating Glioblastoma Invasion

Project description:Introduction: Glioblastoma (GBM) invasion studies have focused on coding genes, while few studies evaluate long non-coding RNAs (lncRNAs), transcripts without protein-coding potential, for role in GBM invasion. We leveraged CRISPR-interference (CRISPRi) to evaluate invasive function of GBM-associated lncRNAs in an unbiased functional screen, characterizing and exploring the mechanism of identified candidates. Methods: We implemented a CRISPRi lncRNA loss-of-function screen evaluating association of lncRNA knockdown (KD) with invasion capacity in Matrigel. Top screen candidates were validated using CRISPRi and oligonucleotide(ASO)-mediated knockdown in three tumor lines. Clinical relevance of candidates was assessed via The Cancer Genome Atlas(TCGA) and Genotype-Tissue Expression(GTEx) survival analysis. Mediators of lncRNA effect were identified via differential expression analysis following lncRNA KD and assessed for tumor invasion using knockdown and rescue experiments. Results: Forty-eight lncRNAs were significantly associated with 33-83% decrease in invasion (p<0.01) upon knockdown. The top candidate, LINC03045, identified from effect size and p-value, demonstrated 82.7% decrease in tumor cell invasion upon knockdown, while LINC03045 expression was significantly associated with patient survival and tumor grade(p<0.0001). RNAseq analysis of LINC03045 knockdown revealed that WASF3, previously implicated in tumor invasion studies, was highly correlated with lncRNA expression, while WASF3 KD was associated with significant decrease in invasion. Finally, WASF3 overexpression demonstrated rescue of invasive function lost with LINC03045 KD. Conclusion: CRISPRi screening identified LINC03045, a previously unannotated lncRNA, as critical to GBM invasion. Gene expression is significantly associated with tumor grade and survival. RNA-seq and mechanistic studies suggest that this novel lncRNA may regulate invasion via WASF3.

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