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Activation induced cytidine deaminase (AID) in germinal center B cells introduces somatic DNA mutations in transcribed immunoglobulin genes to increase antibody diversity. Ectopic expression of AID coupled with selection has been successfully employed to develop proteins with desirable properties. However, this process is laborious and time consuming because many rounds of selection are typically required to isolate the target proteins. AID expression can also adversely affect cell viability due to off target mutagenesis. Here we compared stable and transient expression of AID mutants with different catalytic activities to determine conditions for maximum accumulation of mutations with minimal toxicity. We find that transient (3–5 days) expression of an AID upmutant in the presence of selection pressure could induce a high rate of mutagenesis in reporter genes without affecting cells growth and expansion. Our findings may help improve protein evolution by ectopic expression of AID and other enzymes that can induce DNA mutations.

Transient AID expression for in situ mutagenesis with improved cellular fitness