Engineered proteins that enable site-specific protein and genome modification

Abstract

Dissertation Advisor: David R. Liu Christopher John Podracky

Engineered proteins that enable site-specific protein and genome modification Abstract

Protein engineering, whether by rational design or directed evolution, is a means of generating useful biotechnological and therapeutic tools. First, I describe the application of directed evolution to the bacterial transpeptidase sortase A. Epitope-specific enzymes like sortase A are powerful tools for site-specific protein modification, but generally require genetic manipulation of the target protein to introduce the epitope that they recognize. I describe the directed evolution of sortase A to recognize the LMVGG sequence in endogenous the Alzheimer’s disease-associated amyloid beta-protein (Aβ). Using a yeast display selection for covalent bond formation, we evolved a sortase variant that prefers LMVGG substrates from a starting enzyme that prefers LPESG substrates, resulting in a >1,400-fold change in substrate preference. We used this evolved sortase to label endogenous Aβ in human cerebrospinal fluid, enabling detection of Aβ with sensitivities rivaling those of commercial assays. The evolved sortase can conjugate a hydrophilic peptide to Aβ42, greatly impeding the ability of the resulting protein to aggregate into higher-order structures. These results demonstrate laboratory evolution of epitope-specific enzymes towards endogenous targets as a strategy for site-specific protein modification without target gene manipulation and enable potential future applications of sortase-mediated labeling of Aβ peptides. Next, I described efforts to use directed evolution to alter the specificity of site-specific recombinases (SSRs), a group of enzymes that can perform a variety of predictable and efficient DNA manipulations, but whose broader utility is limited by their requirement for their cognate substrates. This work led me to apply another class of engineered proteins, prime editors, for the programmable insertion of SSR recognition sequences and the development of twin prime editing (twinPE), a method for the programmable replacement or excision of DNA sequence at endogenous human genomic sites without double-strand DNA breaks. TwinPE uses a prime editor (PE) protein and two prime editing guide RNAs (pegRNAs) that template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, resulting in the replacement of endogenous DNA sequence between the PE-induced nick sites with pegRNA-encoded sequences. We show that twinPE can perform precise deletions of up to 780 bp and precise replacements of genomic DNA sequence with new sequences of up to 108 bp. By combining single or multiplexed twinPE with SSRs, either in separate steps or in a single step, we demonstrate targeted integration of gene-sized DNA plasmids at safe-harbor loci including AAVS1, CCR5, and ALB. These results, to our knowledge, represent the first targeted insertion of gene-sized DNA sequences into previously unmodified human cells without requiring double strand breaks or homology directed repair. TwinPE can also mediate a 39-kb inversion at IDS that could be used to correct a common Hunter syndrome allele. TwinPE substantially expands genome editing capabilities without requiring double-strand DNA breaks and, in combination with other genome editing tools, enables the correction or complementation of complex pathogenic allele variants in human cells.