Enabling Precision Editing with CRISPR Hybrid RNA-DNA Guided Cas9

The RNA-guided CRISPR-Cas system is a promising technology with applications in a variety of fields including the development of human therapeutics. However, in order to successfully deploy genome editing for clinical applications it is critical to design strategies that maximize on-target activity while minimizing off-target editing. Previously, we developed a comprehensive and robust two-step specificity analysis pipeline that involves (1) identifying off-target sites with our biochemical SITE-Seq^® assay and (2) inspecting those sites for editing in cell-based experiments with targeted deep sequencing. We have now applied this pipeline to develop CRISPR hybrid RNA-DNA (chRDNA) guides to attenuate off-target cleavage. Here, we will discuss our chRDNA guide selection workflow which allows us to develop chRDNA guides that exhibit editing efficiencies similar to their crRNA counterparts in human primary T cells and with significantly reduced off-target activity. chRDNA guides enable a high activity, high specificity alternative to the standard system for use in therapeutic applications where the utmost precision is required.