Elucidation of Context Dependent Factors Influencing CRISPR-Cas Activity
Since being developed as a tool in 2012, CRISPR-Cas9 and other CRISPR systems have revolutionized the way we manipulate biology. CRISPR systems broadly rely on a programmable guide RNA (gRNA) to enable targeted nuclease activity. Additionally, the ability to target a protein to a specific sequence of DNA has enabled a myriad of applications, including transcriptional silencing and/or activation, single base editing and targeted transposase activity. However, there remains a gap in knowledge as to what factors influence the activity of these systems. The gRNA sequence has been shown to at least partially predict activity but the mechanism behind this is not well understood. In addition, other contextual factors such as DNA repair, target accessibility, and others may play a role. Here, we present factors that influence Cas9 activity in E. coli and other organisms. Additionally, we find that gRNA sequence influences activity in large part by determining how fast Cas9 finds the target site. Together, the work presented improves our understanding of Cas9 and could lead to better gRNA prediction algorithms and new routes to improve Cas9 on-target activity.
Moreb, Eirik Adim (2021). Elucidation of Context Dependent Factors Influencing CRISPR-Cas Activity. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/24361.
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