Project description:To identify mutations that occurred in the nuclear and mitochondrial DNA of the yeast subjected to mtDNA base editing or Mito-BE screen, we performed whole-genome sequencing of cultured yeast cells after isolation of mitochondrial DNA.
Project description:Genome-wide knockout or knockdown screens have become powerful tools for the investigation of genotype-to-phenotype relationships. In bacteria, these screens commonly rely on transcriptional repression by dCas9, gene knockouts through Cas9 editing or random transposon mutagenesis, but depending on the technique, suffer from incomplete gene silencing, low editing efficiencies or they require massive library sizes. Here, we take a distinct approach with base editing to introduce premature stop codons or mutate start codons in Escherichia coli using a ScCas9 nickase derived base editor (ScBE3) that exhibits flexible PAM recognition. We then derive guide design rules by applying machine learning to a gene essentiality screen conducted in E. coli. For further improvement, we combined base-editing with Cas9-induced cleavage of the unedited cell fraction. The efficiency of this dual system was validated through a screen of conditionally essential E. coli genes. This improved setup that decouples the gene editing from the screening leads to more efficient guide depletion and confirmed previously published conditionally essential genes. Overall, base editing represents a useful tool for genome-wide knockout screens in bacteria and will eventually enable genome-wide knockout screens in a broader range of bacterial species to study their diverse genetics.
Project description:Donated human MII oocytes and zygotes were injected with ABE mRNA or RNP, or Cas9 RNP targeting indicated genes. The purpose of the experiment is to study DNA repair after base editing in preimplantation human embryos. Key findings are a lack of chromosomal aneuploidies in base editing samples, contrasting with the outcomes after Cas9 cleavage at the same sites. Off target base editing is highly gRNA dependent. Base editing is efficient, and lacks the genotoxicity of Cas9.
Project description:We developed a CRISPR/Cas9-mediated base-editing screen to functionally screen endogenous proteins. We screened SPEN, a key factor in X chromosome inactivation, for loss-of-function mutations. Some of the screen hits were mutations of serine residues that could be potential phosphorylation sites (by prediction) and could serve as regulatory sites for the function and/or structure of SPEN. To follow up on our observations, we analyzed phosphorylation sites of SPEN in mouse embryonic stem cells, in which both X chromosomes are active (“noDox” sample), and in cells in which X chromosome inactivation was artificially induced by Xist expression (“Dox” sample).
Project description:Insufficient functional T cell persistence impedes therapeutic success of chimeric antigen receptor (“CAR”) therapies. Here, we performed a CAR-adapted base editing screen of PIK3CD, a key regulator of T cell function, metabolism, and fate. We identified point mutations that beneficially modulate CAR T cell profiles in 4-1BBz and 28z CAR T cells, respectively. Remarkably, point mutations with differing effects on PI3Kδ signaling activity were advantageous in distinct CAR contexts: The PI3Kδ-activating mutation E81K enhanced proliferation, metabolic fitness and effector function in 4-1BBz CARs, promoting long-term functional persistence and enhanced therapeutic efficacy in vivo. Conversely, the PI3Kδ-attenuating mutation L32P improved T cell memory formation and functionality in 28z CAR T cells. Together, our approach of Rational Optimization of Activation-dependent Signaling via Targeted Allelic Reprogramming (ROADSTAR) illustrates the importance of CAR design-specific fine-tuning of tailoring intrinsic T cell signaling and demonstrates the potential of base editing for next-generation cellular therapies.