OmniChange: The Sequence Independent Method for Simultaneous Site-Saturation of Five Codons

Abstract

Focused mutant library generation methods have been developed to improve mainly “localizable” enzyme properties such as activity and selectivity. Current multi-site saturation methods are restricted by the gene sequence, require subsequent PCR steps and/or additional enzymatic modifications. Here we report, a multiple site saturation mutagenesis method, OmniChange, which simultaneously and efficiently saturates five independent codons. As proof of principle, five chemically cleaved DNA fragments, each carrying one NNK-degenerated codon, were generated and assembled to full gene length in a one-pot-reaction without additional PCR-amplification or use of restriction enzymes or ligases. Sequencing revealed the presence of up to 27 different codons at individual positions, corresponding to 84.4% of the theoretical diversity offered by NNK-degeneration. OmniChange is absolutely sequence independent, does not require a minimal distance between mutated codons and can be accomplished within a day.

Phosphorothioate-Based DNA Recombination: An Enzyme-Free Method for the Combinatorial Assembly of Multiple DNA Fragments

“…PTRec is not limited by the number of genes to be recombined and enables immediate and efficient cloning of the resulting chimeric library. PTRec was developed after extensive optimization of DNA cleavage and hybridization conditions using the recently published phosphorothioate-based ligase-independent gene cloning (PLICing) method (24) and the OmniChange method (25), which were developed as a DNA fusion technology for cloning random mutant libraries of single genes and a method for the simultaneous site saturation mutagenesis of five codons in a single protein, respectively.…”

To get what we aim for – progress in diversity generation methods

“…Focused mutagenesis is mainly used in semi-rational protein design experiments [14] to improve localized properties such as activity [9,[19][20][21], selectivity [22][23][24] and, less-frequently, thermal resistance [15,25,26]. The prerequisite is a crystal structure or a homology model to identify the 'beneficial' residues that are subsequently randomized [27].…”

“…TrimerDimer [40] SILM [42] OmniChange [9] SySM [46] Amber Codon [51] Number of targeted aa-positions Table 2. Performance criteria of random mutagenesis methods.…”

“…• TrimerDimer [38] • SILM [40] • OmniChange [9] • SySM [44] • Amber Codon [49] • MCST [55] • dITP-epPCR [60] • POE-PCR [61] • MegAnneal [58] • TRINS [54] • epRCA [56] • SeSaM-III [59] • TMGS-PCR [92] • USERec [96] • GoldenGate Shuffling [95] • PTRec [91] • Integron [94] Covered only 48 clones revealed 66-84% coverage of all possible codons at all five targeted positions (up to 27 out of 32 possible codons). OmniChange fulfills all the requirements in terms of robustness and simplicity with respect to becoming a successful method for focused multisite saturation mutagenesis.…”

“…OmniChange [9] solved the primer length challenges and allows multi site saturation of positions all over the protein. SILM [42] and especially the TrimerDimer method [40] have paved the way for minimizing screening efforts by showing new opportunities for avoiding stop and redundant codons.…”