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ABSTRACT: Mismatch target library for VEGFA (T3)

PROVIDER: PRJNA1006350 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR25663137.fastq.gz	Fastqsanger.gz
	SRR25663138.fastq.gz	Fastqsanger.gz
	SRR25663139.fastq.gz	Fastqsanger.gz

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Project description:We performed an original protocol called synthetic STARR-seq (PMID: 30427832) in which a library of putative response elements are tested for their capacity to bind thyroid hormone (T3) nuclear receptors and convey T3 transactivation More than 10 000 putative T3 response elements (T3RE) were cloned in the hSTARR-seq-ORI vector with a minimal promoter, in the 3' end of the transcribed sequence. These are variant of the consensus T3RE (5'NGGTCANNNNRGGNNA3') Therefore the most active T3RE are over-represented in the RNA of cells transfected with the plasmid library and treated with T3.

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Project description:Purpose: We report expression of a new Cas13 enzyme in retinal cells and evaluate its efficacy in targeting the VEGFA mRNA to establish safety and efficacy of the enzyme for anti-VEGFA therapy. Methods: Mammalian cells (HEK293FT) or mouse retina were treated with shRNA, CasRx or Cas13bt3 carrying VEGFA targeting sgRNA. Total RNA was collected for RNA seqeuencing to study VEGFA mRNA expression and well as off-target genes. Results: Significant knockdown of VEGFA mRNA was observed with no effect on predicted off-target genes in mammalina cells and mouse retina. No loss of mouse VEGFA was also detected. Conclusions: AAV2.7m8 can be used for retina-wide transduction, and Cas13bt3 may be a potential new tool for control of VEGFA.

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