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. Author manuscript; available in PMC: 2021 Jul 7.
Published in final edited form as: Structure. 2020 Apr 30;28(7):760–775.e8. doi: 10.1016/j.str.2020.04.009

Figure 3: Comparison of engineering on the I-OnuI vs. the thermostabilized eOnuTherm meganuclease scaffolds.

Figure 3:

(a) List of target sites for the series of libraries designed, generated, and screened on both the I-OnuI and eOnuTherm protein scaffolds toward the desired bCtxA sequence. Library 04 (Lib04) produced no active variants on the I-OnuI scaffold (marked “Failed” with a red X) but was successful on the eOnuTherm scaffold. Additional libraries were screened on the I-OnuI scaffold (toward changes on the right half-site of the DNA target), but selections failed again at Library 08 (Lib08, also marked “Failed”). In contrast, the thermostabilized eOnuTherm scaffold was successfully re-engineered to target the bCtxA DNA sequence after the step-wise generation of seven libraries, with no selection steps failing to produce active variants. (b) Locations of 13 mutations introduced across the eOnuTherm protein to create the bCtxA-targeting meganuclease (eOnuTherm-bCtxA) are illustrated with red spheres on the alpha carbons of the mutated sidechains. (c) Expression and activity of the re-engineered eOnuTherm-bCtxA meganuclease on the surface of yeast using flow cytometry, as previously described in Figure 1, ‘Methods’ and Supplementary Figure S1. (d) In vitro analysis of DNA cleavage activity with eOnuTherm-bCtxA meganuclease released from the surface of yeast and no physical tethering of the DNA substrate. (e) Circular dichroism (CD) thermal denaturation for the analysis of protein thermostability. The original eOnuTherm meganuclease (blue) unfolds at 56°C, while the final re-engineered eOnuTherm-bCtxA protein (red) shows a reduced melting temperature of 46°C. The best-performing active clone from Lib07 on the I-OnuI scaffold (slate) melts at an even lower temperature of 42°C. The thirteen mutations required to generate the final eOnuTherm-bCtxA-targeting enzyme were grafted onto the wild type I-OnuI scaffold and resulted in a protein with a Tm value of 41°C (pink), which represents a 4° reduction relative to wild-type I-OnuI (Figure 1e, 2c).