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Multiplexed inactivation of endogenous genes by base-editor-mediated multi-stop system

ABSTRACT: Multiplexed inactivation of endogenous genes by base-editor-mediated multi-stop system

PROVIDER: PRJNA779867 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR16925452_1.fastq.gz	Fastqsanger.gz
	SRR16925452_2.fastq.gz	Fastqsanger.gz
	SRR16925453_1.fastq.gz	Fastqsanger.gz
	SRR16925453_2.fastq.gz	Fastqsanger.gz
	SRR16925454_1.fastq.gz	Fastqsanger.gz

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Multiplexed inactivation of endogenous genes by base-editor-mediated multi-stop system

Project description:Base editor is also a powerful tool to introduce early stop codons to simultaneously silence multiple genes to study family gene function in embryo development. Here, we identified the highest efficiency base editing system (hA3A-eBE-Y130F) with few sgRNA independent off-target sites among six well-established base editors in mouse embryo. Further, we developed a Multi-Stop system to simultaneously knock-out all Ten-eleven translocation (TET) family of dioxygenase genes (Tet1, Tet2 and Tet3) in zygotic genome leads embryo die before 10.5 days of gestation. Moreover, we showed that inactivation of three DNA methyltransferases genes (Dnmt1, Dnmt3a and Dnmt3b) in zygote leads to gastrulation failure. We also obtained multiple TET and DNMT family genes (6KO: Dnmt1/3a/3b and Tet1/2/3; 5KO: Dnmt3a/3b and Tet1/2/3; 4KO: Dnmt1 and Tet1/2/3) deactivated mouse embryos in one step, which indicated that Dnmt1 or Dnmt3a/b is indispensable for mouse gastrulation.

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Base-editor-mediated multiplexed inactivation of DNA methyltransferases reveals essential roles of miRNAs in mouse gastrulation

Project description:Here, we first establish an easy Multi-Stop system to simultaneously inactivate genes involved in DNA methylation and demethylation in zygotes through introduction of the stop codon by hA3A-eBE-Y130F-mediated base editor (BE). While Multi-Stop-derived Dnmt-null embryos display embryonic lethal due to gastrulation failure. Moreover, mutation combinations between Tet and Dnmt families show severe embryonic lethal and Dnmt1 or Dnmt3a/3b is indispensable for mouse gastrulation. Then WGBS and RNA-seq analysis of different mutant embryos reveals genes jointly maintained by Dnmt1 and Dnmt3a/3b that are critical for gastrulation.

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Base-editor-mediated multiplexed inactivation of DNA methyltransferases reveals essential roles of miRNAs in mouse gastrulation

Project description:Base-editor-mediated multiplexed inactivation of DNA methyltransferases reveals essential roles of miRNAs in mouse gastrulation

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Base-editor-mediated multiplexed inactivation of DNA methyltransferases reveals essential roles of miRNAs in mouse gastrulation

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Base-editor-mediated multiplexed inactivation of DNA methyltransferases reveals essential roles of miRNAs in mouse gastrulation

Project description:Here, we establish a system to simultaneously inactivate Dnmts in one step through screening for base editors that can efficiently introduce a stop codon endogenously. Dnmt-null embryos display primitive streak elongation failure at E7.5. Interestingly, although DNA methylation is absent, gastrulation-related pathways are down-regulated in Dnmt-null embryos. Moreover, DNMT1 or DNMT3A/3B are indispensable for gastrulation and their functions are independent of TET proteins. Hypermethylation can be sustained by either DNMT1 or DNMT3A/3B at some promoters, which are mainly related to miRNAs. Strikingly, majority of hypermethylated promoter-related miRNAs are overexpressed and located at the Dlk1-Dio3 imprinted region. The predicted targets of these miRNAs tend to be down-regulated and enriched in the gastrulation-related pathways in Dnmt-null embryos. Finally, introduction of a single mutant allele of six miRNAs partially restores primitive streak elongation in Dnmt-null embryos.

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Project description:Multiplexed inactivation of endogenous genes by base-editor-mediated multi-stop system

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