The emergence of CRISPR-based genome-editing therapeutics, a base-editing technology to modify the target genome at the single base pair level, has opened new frontiers for the cure of pathogenic genetic disorders that are caused by single nucleotide polymorphisms (SNPs). The most recent base-editing tool, Adenine Base Editor (ABE), can carry out the chemical transformation of an adenine(A) to a guanine(G) at the target site. ABE was developed using the most advanced strategy for protein engineering - directed evolution. It consists of an impaired Cas9 protein fused to a tRNA-editing enzyme, TadA, which was evolved to edit single-stranded DNA, using directed evolution. The Cas9 serves as a guide to the desired genomic site and the mutant TadA protein carries out the chemical reaction on the target DNA site.<br/>Currently, the lack of natural enzymes that can successfully mutate the genomic DNA at the single base level is seriously impeding the development of base-editor therapy. Although directed evolution can be used to mutate more tRNA-editing enzymes to generate more CRISPR-base editors, it has some limitations.<br/>To reduce the experimental effort associated with directed evolution and to explore the sequence space encoded by mutating multiple positions simultaneously, we employ computational design strategies. All-atom, explicit-solvent, molecular dynamics (MD) simulations and umbrella sampling calculations of the various DNA-TadA mutant complexes were performed on GPUs using the CUDA version of PMEMD in AMBER. These GPU-accelerated simulations help us decipher the role of the DNA-protein binding interactions for the various evolved generations of the TadA protein. We validate our hypotheses and the design paradigm on the ABE protein, demonstrating that computational methods can indeed help to rationalize the various mutations in the ABE base-editor. We plan on generalizing this engineering approach and aiding the discovery of more genome editors in conjunction with canonical directed evolution.

Rational engineering of CRISPR-based genome editors using computational methods

2019 Fall National Meeting

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