Expanding the utility of precision genome editing

Abstract

The advent of CRISPR-Cas systems has sparked a renaissance in genome editing. The ability to precisely, accurately, and efficiently edit the genome of living cells has been a longstanding aspiration of the life sciences. The double-strand breaks (DSBs) generated by programmable nucleases such as CRISPR-Cas9 result primarily in a stochastic mixture of base insertions and deletions at the target site. These chaotic repair events are useful for gene disruption, but lack the capability to enable precise DNA edits. Furthermore, recent studies have shown that DSBs may result in large cellular perturbations such as undesired translocations, chromothripsis, large deletions, and p53 activation. The challenges of using DSBs to generate precise gene edits in most cell types and the undesired consequences of inducing cellular DSBs has highlighted the need to develop precision genome editing methods that do not require double-strand DNA break intermediates. Base editors are genome editing technologies that leverage the ability of CRISPR-Cas proteins to find specific sequences of DNA as directed by a modular guide RNA sequence. Base editors consist of a programmable DNA binding protein such as a catalytically impaired Cas protein or a TALE repeat array, as well as a deaminase enzyme that catalyzes the conversion of the targeted DNA base to a different base. The DNA-binding protein localizes the deaminase enzyme to a target sequence, thus enabling the deaminase to perform chemistry directly on the genome of living cells. The two major classes of base editors are cytosine and adenine base editors, which enable the conversion of C•G to T•A base pairs or A•T to G•C base pairs, respectively. Both classes of base editors are widely used for biological research and for potential clinical applications. In this thesis, I present four efforts to expand the potential and applicability of cytosine and adenine base editors. In chapter two, I will discuss the use of a small bacteriophage Mu Gam protein to enhance the product purity of cytosine base editors. In chapter three, I will describe efforts to expand the targeting scope of base editors by engineering the Cas9 domain to accept other protospacer adjacent motifs. Through this study, we observed that adenine base editors had lowered editing efficiencies when using other Cas protein orthologs. This limitation of original adenine base editors led us to hypothesize that the adenosine deaminase domain could be further optimized to increase activity and compatibility with Cas protein variants. To address this limitation, I describe a phage assisted continuous evolution (PACE) effort in chapter four in which we used PACE to evolve new editor variants that were ~1,000-fold improved in deamination kinetics, resulting in drastically enhanced adenine base editing activity in living cells. Finally, in chapter five I will describe efforts to use small molecules to establish sensitive and rapid control over genome editing activity. We demonstrate that pomalidomide can promote degradation of modified base editors to attenuate editing activity. Alternatively, pomalidomide can drive the destruction of an anti-CRISPR protein to enable activation of genome editing. Collectively, these developments in base editing increase the capabilities of precision genome editing and the potential application of these tools in biological research, medicine, and agriculture.