1. We improved MAGESTIC (Roy et al. 2018) by optimizing guide RNA and Cas9 expression to boost the efficiency of homology directed repair (HDR). We benchmarked donor recruitment against alternative HDR enhancement strategies, and combined those into a single, superior editing method termed (Roy et al., in preparation).
2. We developed methods to investigate the efficiency and fidelity of precision editing, including a fast, simple, cheap, and scalable method that produces sequencing-ready libraries directly from yeast cultures (Vonesch et al. 2021). We performed genome sequencing of thousands of edited strains and confirmed that the vast majority of clones carried the desired variants without off-target effects. We found that the likelihood of erroneous on-target structural variants was dependent on genome context, and constructed machine-learning models to predict these “hard-to-edit” regions, facilitating the development of improved editing methods (Li et al., in preparation).
3. We constructed variant pools and isolated thousands of strains, each with a different variant. We optimized phenotyping and analytic pipelines towards maximizing sensitivity for subtle effects. We profiled fitness across chemical, drug and nutritional perturbations, to investigate how variants are active under different environmental conditions. We found that most genes have not been linked to growth in these conditions in previous knockout screens. Their protein products show physical interactions with those of other genes implicated in these traits (Vonesch et al., in preparation). Many natural variants show genetic interactions with genes implied in the same trait by the KO screen. We used an improved MAGESTIC version to dissect QTL down to their causative nucleotide variants (Roy et al., in preparation).
4. Many SNPs only have an effect in the presence of another SNP. To enable systematic discovery of such genetic interactions, we developed a new method that allows single cells to obtain multiple edits. A key achievement was the development of a barcoding system capable of linking the barcodes from multiple rounds of editing, allowing the edit combinations to be read out by short-read sequencing (Roy et al., manuscript in preparation).
5. We introduced several novel methods that allow characterization of 1,000s of CRISPR perturbation effects within a single experiment. One of these methods, termed image-enabled cell sorting (Schraivogel et al. 2022), allows isolation of cells according to information from microscopic images at speeds up to 15,000 cells per second. This method will enable new types of experimental strategies and is compatible across organisms, from yeast to mammalian cells. The other method is a targeted, single-cell RNA-seq assay for the massively parallel molecular phenotyping of cells carrying genetic perturbations. We obtained rich molecular phenotyping information on the expression of ~200 genes across ~1,000 genetic perturbations (Schraivogel, Gschwind et al. 2020), and are now using it to assign functions to disease associated genetic variants (ongoing work).
