Abstract

The use of synthetic CRISPR guide RNAs (gRNA) for generating functional gene knockouts provides distinct advantages over other screening strategies utilized in research. In the past several years, however, pooled lentiviral CRISPR libraries have been the method of choice for high-throughput screening. While this system still has its uses for certain biological questions, disadvantages include a high data analysis burden, safety concerns, and low signal-to-noise ratio. Here, we investigate and compare many variables and design parameters associated with non-viral CRISPR libraries across different cell types, including iPSCs. These variables include delivery of multiple gRNAs for a given gene into a single transfection (arrayed-by-gene) vs. delivery of a single gRNA per transfection (arrayed-by-guide). We also place a focus on defining spacing rules for arrayed-by-gene gRNAs to encourage out-of-frame deletions. Based on this work, we have developed a pipeline with specific parameters (e.g., off-target score cut-off, exonic location within the gene, and guide spacing) to design multiple gRNAs for each gene, supporting CRISPR screening projects. Here, we present our screening workflow for delivering the gRNAs and analyzing the editing efficiency in research applications using a 95 gene library involved in DNA repair. Additionally, CRISPR-based homology-directed repair (HDR) is an invaluable tool for research screening projects. We previously reported on optimized HDR design rules and the development of the Alt-R HDR Design Tool, a novel bioinformatics tool that has been trained on empirical data for the design of HDR donor templates in an easy-to-use, open-access web format (Schubert et al., 2021). Here, we will also present further advancements in the design of HDR experiments, as well as the use of novel HDR enhancers to achieve high HDR efficiency of small and large insertions in immortalized cells, primary cells, and iPSCs.