Harnessing the Peculiarities of the Cas9 Reaction Sequence to Enhance Genome Editing

Abstract

Targeted manipulation of the mammalian genome has been revolutionized by the RNA-guided nuclease, Cas9, from the S. pyogenes clustered regularly interspaced short palindromic repeats (CRISPR) system. A biologist can now target virtually any region of the genome to induce mutations, activate or repress transcription, and compact or decompact chromatin. Cas9 activities can be highly multiplexed within a single cell, providing an unprecedented opportunity to manipulate the mammalian genome and to program cellular behaviors. My studies have focused on characterizing and engineering of Cas9 to the understand how our cells interact with it, how to make genome editing more efficient, and how to utilize Cas9 as an interface between the biologist and the genome to program complex cellular behaviors. My biochemical studies were focused on the strange properties the foreign nuclease displays. Specifically, Cas9 stably binds DNA-products, effectively converting it to a single-turnover nuclease. I found this to be the rate limiting step to a genome edit, whereby Cas9 must dissociate from the broken genome for mutagenesis to occur. Cas9 can be removed from the genome by translocating RNA polymerases, or by fusing a cell cycle regulated degradation motif directly to the nuclease to promote its destabilization. These studies have uncovered the essential role that endogenous cellular processes play in affecting the efficiencies of a genome edit. Furthermore, degron associated degradation of Cas9 indicated that Cas9 can be modified to be recognized by the cell. Increasing the efficiency of DNA targeting by Cas9 is a requisite to utilize the nuclease as powerful cellular programming tool. However, there is no ability to control the sequence of Cas9 activities. Considering biological processes exist as sequences of events, the ordering of Cas9 mediated genetic events is required to recreate complex biology outside of the body and to program novel biology in mammalian cells. Working towards this concept, I focused on engineering of the guide RNA rather than Cas9 as, as the guide directs Cas9 activities. By converting guide RNAs into switches (“pGuides”) that exist in the off-start and convert each other into the on-state, cascades of pGuides enabled sequentially regulated Cas9 activities. The cascades work to program up to four sequential mutagenic events in proof of concept experiments. Interestingly, the pGuides also could be repurposed as a means to mutate a site over a 150 day period. This demonstration is a proof of principle for long-term molecular barcoding via Cas9, and increases the capacity of barcoding by 20 fold compared to existing technologies. In summary, my studies uncover mechanisms of genome editing and demonstrate the versatility of the CRISPR/Cas9 system.