Determinants of base editor efficacy are position-specific
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ABSTRACT:
PROVIDER: PRJEB44304 | ENA |
REPOSITORIES: ENA
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Project description:Proteins are key molecular players in a cell, and their abundance is extensively regulated not just at the level of gene expression but also post-transcriptionally. Here, we describe a genetic screen in yeast that enables systematic characterization of how protein abundance regulation is encoded in the genome. The screen combines a CRISPR/Cas9 base editor to introduce point mutations with fluorescent tagging of endogenous proteins to facilitate a flow-cytometric readout. We first benchmarked base editor performance in yeast with individual gRNAs as well as in positive and negative selection screens. We then examined the effects of 16,452 genetic perturbations on the abundance of eleven proteins representing a variety of cellular functions. We uncovered hundreds of regulatory relationships, including a novel link between the GAPDH isoenzymes Tdh1/2/3 and the Ras/PKA pathway. Many of the identified regulators are specific to one of the eleven proteins, but we also found genes that, upon perturbation, affected the abundance of most of the tested proteins. While the more specific regulators usually act transcriptionally, broad regulators often have roles in protein translation. Overall, our novel screening approach provides unprecedented insights into the components, scale and connectedness of the protein regulatory network.
2022-11-07 | PXD029363 | Pride
Project description:Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show Efficacy of cytosine and adenine base editors in modelingCTNNB1hot-spot mutations in hepatocyte organoids. Next, we use C>T base editors to insert nonsense mutations inPTENin endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug screening assays on organoids harboring eitherPTENorPTENandPIK3CAmutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C>T and A>G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.
2023-07-30 | GSE236490 | GEO
Project description:Transcriptome wide edits after split-engineered base editor treatment
| PRJNA750724 | ENA
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.
2023-04-19 | GSE188658 | GEO
Project description:Recent optimization of CRISPR/Cas9-mediated genome engineering has resulted in the development of base editors that can efficiently mediate C>T and A>G transitions. Combining these genome engineering tools with human adult stem cell (ASC)-derived organoid technology holds promise for disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived hepatocyte, endometrial and intestinal organoids. First, using conventional and evolved Cas9-variants, we show efficacy of both cytosine and adenine base editors and use them to model four hot-spot point mutations in CTNNB1 in hepatocyte organoids. Next, we apply C>T base editors in endometrial organoids to insert nonsense mutations in PTEN and demonstrate tumorigenicity even in the heterozygous state. Furthermore, we use cytosine base editors for simultaneous oncogene activation (PIK3CA) and tumor-suppressor inactivation (APC and TP53). To increase the flexibility of base editor multiplexing, we then combine SpCas9 and SaCas9 base editors for simultaneous C>T and A>G editing at individual target sites. Finally, we show the power of base editor multiplexing by modeling colorectal tumorigenesis in a single step by simultaneously transfecting sgRNA’s targeting four cancer genes.
Seven clonal organoid lines and one bulk wild-type control sample were paired-end whole-genome sequenced using the Illumina Novaseq 6000 system. We sequenced four clonal intestinal organoid lines harbouring engineered TP53 and FBXW7 mutations as well as three lines targeted for oncogenic APC/TP53/PIK3CA/SMAD4 mutations. This WGS showed, as previously reported, a genome-wide increase in C>T mutations due to C>T base editor off-target activity, which is not enriched in predicted off-target regions based on the sgRNA sequences. Furthermore, we confirmed the absence of editing-induced driver mutations and lack of off-target mutational hotspots created by the genomic engineering.
| EGAS00001006886 | EGA
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.
2023-04-23 | GSE162903 | GEO
Project description:Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here, we describe “signaling-to-transcription network” mapping through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally-resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of PHLPP1 which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.
2024-02-26 | GSE244164 | GEO
Project description:Eliminating genome-wide and transcriptome-wide off-target mutations by base editor
| PRJNA690814 | ENA
Project description:We report transcriptome wide edits comparison between split-engineered base editors and intact base editors. Our results show that, split-engineered base editors show backgound levels of unique C>U edits when compared to intact base editors.
2021-10-18 | GSE181109 | GEO
Project description:Conjugation of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) for programmable C-to-T editing, which holds potentials in clinical applications, but suffers from off-target (OT) mutations. By taking advantage of a cleavable deoxycytidine deaminase inhibitor (dCDI) domain, a transformer BE (tBE) system is developed to induce efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, tBE remains inactive at OT sites with the fusion of a cleavable dCDI, thus eliminating unintended mutations. Only when binding at on-target sites, tBE is transformed to cleave off the dCDI domain and catalyzes targeted deamination for precise base changes. After delivery into mice via a dual-AAV system, tBE created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9 level, which resulted in ~30-40% decrease of total cholesterol. Together, the development of tBE establishes a highly-precise base editing system and its in vivo efficacy envisions potential therapeutic applications.
2021-03-12 | GSE164477 | GEO