Evolved base editors with high precision and minimized off-target activity by a novel continuous directed evolution system in mammalian cells

Continuous directed evolution of CRISPR-Cas system or base editors has been successful in bacteria cells, but not yet in mammalian cells. Here, we report the development of a Continuous Directed Evolution system in Mammalian cells (CDEM). CDEM enables the BE evolution in a full-length manner with Cas9 nickase rather than a split BE with dead Cas9 as reported by bacteria-based evolution system. We next harnessed CDEM to evolve the deaminases of cytosine base editor BE3 and adenosine base editor ABEmax and ABE8e using delicate circuits for narrowing or shifting the editing window with the attempt to minimize undesired bystander mutations and to improve the targeting spectrum of the base editors. The evolved cytidine deaminase variants on BE4 architecture showed not only narrowed editing windows, but also higher editing purity and low off-target activity without a trade-off in on-targeting activity in comparison with wild-type BE4. Specifically, N7-BE4 variant outperforms the previously reported YE1-BE4. The evolved ABEmax and ABE8e variants exhibited narrowed or shifted editing windows to different extents, and lower off-target effects. The results illustrate CDEM as a simple but powerful approach, can continuously evolve BEs without size restriction in the mammalian environment. We envision that CDEM could be used to evolve other CRISPR-associated effectors or other biological proteins.