Determinants_of_genome_editing_efficiency_with_Cas9_derived_tools. Determinants_of_genome_editing_efficiency_with_Cas9_derived_tools

Background Our goal for the next years is to install precise edits to human genomes and to evaluate their effect at scale, as well as in different genetic contexts. The toolbox of choice is CRISPR/Cas, which can bring a variety of enzymatic activities to specific locations in the genome. In particular, double strand break followed by homology-directed repair allows insertion of designed donor sequence, base editing directly converts one nucleotide into another through deamination, and prime editing enables the introduction of insertions, deletions, and substitutions through reverse-transcription. Incomplete understanding of editing efficiency reduces the scale at which we can operate, and confounds mutation effect readouts. To counter this, we have previously characterised efficiency determinants of the standard Cas9 protein, as well as C to T base editors. Here, we propose to 1) characterize A to G base editors and prime editors and 2) evaluate their merits for installing point mutations to three essential genes in the HAP1 cell line compared to the gold standard Saturation Genome Editing. Research questions - What is the editing window of A to G base editors, and how does this depend on the sequence context of the targeted base? How pure are the outcomes? - How does donor sequence impact prime editing efficacy at four different targets? Does pegRNA secondary structure help or hinder editing? - Is the readout of mutation effect generated by base editing aligned to ones generated with saturation genome editing? Can we impute the impact of unobserved mutations? - Is the readout of mutation effect generated by prime editing aligned to ones generated with saturation genome editing? Can we impute the impact of unobserved mutations? Proposal - Base editing screens with ~50,000 gRNA library of two A to G and one C to T base editors to understand their sequence specificity of efficacy in HEK293T cells. - Prime editing efficacy screens with a ~2000-sequence pegRNA library to understand the determinants in HEK293T cells - Base editing tiling screens of BRCA1, DDX3X, and BAP1 genes in HAP1 cells using all possible NG-anchored target positions in all exons, two different C to T editors, and two different A to G editors; generating a subset of possible point mutations across all exons. - Prime editing tiling screens of three exons each from BRCA1, DDX3X, and BAP1 genes in HAP1 cells using two alternative pegRNAs for each installed SNV, generating all possible point mutations in a limited number of exons. All screens and sequencing libraries will be done by our lab. The data will help us build comprehensive predictive models on genome editing using different tools and approaches, and to evaluate the most efficient way to scale mutation effect assessment in human cells.