Making the Cut: Modulation of Genome Engineering via Cas9/gRNA Regulation

Manipulation of the genome is a long-sought goal of multiple branches of research and medicine. Discovery of the adaptive immune system evolved in both bacteria and archea, CRISPR/Cas, presented an adaptable system to mediate targetable changes in the genome. Through evolution, a variety of CRISPR/Cas systems, each with their own specificities and effector structures. Many of the CRISPR/Cas systems have been adapted for use in mammalian genome editing. The most characterized system, the CRISPR/Cas9 system, is derived from Streptococcus pyogenes. This system mediates double stranded breaks within the DNA via the effector enzyme, the Cas9 endonuclease. This RNA guided nuclease has been adapted for many roles in genome engineering, but is constantly being modified to improve various outcomes. In this work, we sought to manipulate the function of the CRISPR/Cas9 system through modification of either the Cas9 or the guideRNA structure. Of primary interest is modulation of the DNA repair outcomes following a Cas9 mediated cleavage event; the cellular host machinery is responsible for the repair, and has been recently found to produce similar frequencies of outcomes across replicates and cell lines. While efforts to change these outcomes have been approached, many involve manipulation of repair pathways and cellular perturbations that have a negative impact on viability. Using the addition of degradational motifs fused to the Cas9 enzyme, we established a cell-cycle based regulation of Cas9 degradation. In combination with regulation of Cas9 expression using cell cycle regulated promoters, Cas9 outcomes were modulated towards improved Homology Directed Repair frequencies. Furthermore, these results demonstrated that lower levels of regulated Cas9 can mediate improved outcomes, possibly establishing a higher efficiency system with reduced risk of off-target effects and immune system response. Further regulation of the activity of Cas9 was mediated through the engineering of an ’on/off’ variant of the guideRNA, termed proGuides. We previously established the efficacy of these proGuides to enabled timed, activated Cas9 activity without exogenous signals. These proGuides were further used to enable coordination of the system with use of CRISPR mediated gene activation via Cas9-VPR, a Cas9 fused to transcriptional activators. This system was applied to synthetic activation of myogenesis differentiation, and demonstrated timed, sequential activation of the targets of interest in myogenesis, and enabled increased efficiency in the formation of multinucleate muscle cells. Future work is necessary to further improve these systems, but this work demonstrates the potential of each strategy of CRISPR/Cas9 regulation for genomic engineering.