Directed evolution of genome editing proteins

Abstract

Directed evolution, which applies the principles of Darwinian evolution to a laboratory setting, is a powerful strategy for generating biomolecules with diverse and tailored properties. This technique can be implemented in a highly efficient manner using continuous evolution, which enables the steps of directed evolution to proceed seamlessly over many successive generations with minimal researcher intervention. Phage-assisted continuous evolution (PACE) enables continuous directed evolution in bacteria by mapping the steps of Darwinian evolution onto the bacteriophage life cycle and allows directed evolution to occur on much faster timescales compared to conventional methods. In this thesis, I describe the use of directed evolution, including PACE, for the development robust tools for gene editing applications. First, I describe efforts to increase the targeting scope of the most well-known CRISPR protein, SpCas9. SpCas9 is favored for its robust and reprogrammable activity, as a researcher simply designates 20 nucleotides in a guide RNA sequence to reprogram SpCas9 targeting to a locus of interest through specific RNA:DNA interactions. However, an additional protein:DNA interaction is required prior to RNA:DNA duplex formation: Cas9 must bind the protospacer adjacent motif (PAM), which, for SpCas9, is a specific 5’-NGG-3’ sequence directly 3’ of the protospacer. The PAM is often considered the gatekeeper of Cas9 activity, as PAM binding is the first step of DNA search for the Cas9 sgRNA complex; therefore, a 5’-NGG sequence that occurs only once every ~16 potential SpCas9 target sites is limiting for techniques that require the precise placement of Cas9. We used PACE to expand the targeting scope of SpCas9, leading to a panel of SpCas9 variants that, in total, can access the entirety of the NRN (R=A,G) PAM space. This in principle enables targeting of ~95% of known pathogenic transition mutations by base editing. Next, I describe a brief vignette on using CRISPR associated proteins as RNA responsive molecular switches. RNA is the most dynamic biomolecule in the central dogma, as different cellular states can prompt drastic changes in RNA levels faster than at the protein level. As such, robust tools for the sensing and response to RNA molecules are attractive for the detection and intervention of cellular action in vivo. Current methods for RNA responsive molecular switches, like toehold switches, rely on RNA hybridization and, therefore, lead to varying turn-on depending on the target RNA molecule. In this chapter, I propose and briefly explore using Cas13, an RNA-programmable RNA nuclease, to re-programmably target and respond to cellular RNAs. Finally, I describe progress towards the evolution of CRISPR-associated transposases for the insertion of large, gene-sized DNA into the mammalian genome. The re-programmable insertion of large DNA without the formation of double-stranded breaks is an underdeveloped frontier in precision genome editing. CRISPR-associated transposases are recently described tools that use RNA-guided DNA-binding CRISPR associated proteins to direct Tn7-like transposases to a genomic site of interest. Though these tools are described to work robustly in bacteria, they demonstrate near-zero activity in mammalian cells. In this chapter, I describe progress towards the directed evolution of CRISPR-associated transposases and their characterization in bacterial and mammalian cells.