Transient AID expression for in situ mutagenesis with improved cellular fitness

Abstract

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.

High efficiency CHO cell display-based antibody maturation

“…Based on the two unique features of mouse AID, mAID-plus is the best choice for CHO cell display-based maturation. A recent study by Al-Qaisi et al 26 compared transient and stable transfection (2020) 10:8102 | https://doi.org/10.1038/s41598-020-65044-7…”

Directed evolution in mammalian cells

“…Unfortunately, although originally thought to be targeted to specific genetic loci, it is now clear that AID induces mutations genome-wide in cultured cells, especially at highly transcribed regions. 44,45 Supporting this observation, AID expression is cytotoxic when highly expressed, requiring researchers to artificially moderate expression levels during directed evolution experiments. 44 Thus, the need for truly targeted DNA-damaging enzymes has emerged.…”

“…44,45 Supporting this observation, AID expression is cytotoxic when highly expressed, requiring researchers to artificially moderate expression levels during directed evolution experiments. 44 Thus, the need for truly targeted DNA-damaging enzymes has emerged.…”

“…(b) Activationinduced cytidine deaminase (AID) mutagenizes hypervariable regions of immunoglobulin (Ig) loci, 14 although evidence suggests that significant off-target mutagenesis occurs. 44,45 (c) AID induces point mutations by deaminatingcytosine (C), creating uracil (U) bases that can be unfaithfully repaired or may base-pair with adenine upon replication, resulting in the introduction of C➔T mutations. (d) CRISPR-X targets mutagenesis to user-defined DNA sequences through a targeted guide RNA (gRNA) with a MS2 binding loop that recruits an MS2-AID fusion.…”

Roadmap to Building a Cell: An Evolutionary Approach

“…The fidelity of Φ29 or a similar DNA polymerase could be lowered by using increased Mn 2+ ion concentrations ( Fujii R. et al, 2014 ) or by utilizing exonuclease-deficient variants. Alternatively, increased mutagenesis levels could be achieved by adapting the use of DNA deaminase domains (such as cytidine or adenine deaminases) in vitro either alone ( Al-Qaisi et al, 2018 ) or in fusion with T7 RNA polymerase ( Chen et al, 2019 ; Figure 2D ). Development of these and similar methods will be critical in the diversification of large synthetic genomes.…”