Facile Assembly of Combinatorial Mutagenesis Libraries Using Nicking Mutagenesis.

Combinatorial mutagenesis is a method where multiple user-defined mutations are encoded at defined positions in a sequence. Combinatorial mutagenic libraries can be used in a variety of applications including evaluating fundamental questions about molecular evolution, directed evolution workflows for enzyme engineering, and in better understanding of biological processes like antibody affinity maturation. Here, we show a method of combinatorial mutagenesis utilizing the template-based nicking mutagenesis with several modifications. We show an example for generating a combinatorial library with 14 mutated positions, a total of 16,384 library variants, and a protocol for the generation of large, user-defined combinatorial libraries. The reader can use this protocol to create such libraries in 2 days.

Optimization of multi-site nicking mutagenesis for generation of large, user-defined combinatorial libraries.

Generating combinatorial libraries of specific sets of mutations are essential for addressing protein engineering questions involving contingency in molecular evolution, epistatic relationships between mutations, as well as functional antibody and enzyme engineering. Here we present optimization of a combinatorial mutagenesis method involving template-based nicking mutagenesis, which allows for the generation of libraries with >99% coverage for tens of thousands of user-defined variants. The non-optimized method resulted in low library coverage, which could be rationalized by a model of oligonucleotide annealing bias resulting from the nucleotide mismatch free-energy difference between mutagenic oligo and template. The optimized method mitigated this thermodynamic bias using longer primer sets and faster annealing conditions. Our updated method, applied to two antibody fragments, delivered between 99.0% (32451/32768 library members) to >99.9% coverage (32757/32768) for our desired libraries in 2 days and at an approximate 140-fold sequencing depth of coverage.

Saturation Mutagenesis Genome Engineering of Infective ΦX174 Bacteriophage via Unamplified Oligo Pools and Golden Gate Assembly.

Here we present a novel protocol for the construction of saturation single-site-and massive multisite-mutant libraries of a bacteriophage. We segmented the ΦX174 genome into 14 nontoxic and nonreplicative fragments compatible with Golden Gate assembly. We next used nicking mutagenesis with oligonucleotides prepared from unamplified oligo pools with individual segments as templates to prepare near-comprehensive single-site mutagenesis libraries of genes encoding the F capsid protein (421 amino acids scanned) and G spike protein (172 amino acids scanned). Libraries possessed greater than 99% of all 11 860 programmed mutations. Golden Gate cloning was then used to assemble the complete ΦX174 mutant genome and generate libraries of infective viruses. This protocol will enable reverse genetics experiments for studying viral evolution and, with some modifications, can be applied for engineering therapeutically relevant bacteriophages with larger genomes.

A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis.

Saturation mutagenesis is a fundamental enabling technology for protein engineering and epitope mapping. Nicking mutagenesis (NM) allows the user to rapidly construct libraries of all possible single mutations in a target protein sequence from plasmid DNA in a one-pot procedure. Briefly, one strand of the plasmid DNA is degraded using a nicking restriction endonuclease and exonuclease treatment. Mutagenic primers encoding the desired mutations are annealed to the resulting circular single-stranded DNA, extended with high-fidelity polymerase, and ligated into covalently closed circular DNA by Taq DNA ligase. The heteroduplex DNA is resolved by selective degradation of the template strand. The complementary strand is synthesized and ligated, resulting in a library of mutated covalently closed circular plasmids. It was later shown that because very little primer is used in the procedure, resuspended oligo pools, which normally require amplification before use, can be used directly in the mutagenesis procedure. Because oligo pools can contain tens of thousands of unique oligos, this enables the construction of libraries of tens of thousands of user-defined mutations in a single-pot mutagenesis reaction, which significantly improves the utility of NM as described below. Use of oligo pools afford an economically advantageous approach to mutagenic experiments. First, oligo pool synthesis is much less expensive per nucleotide synthesized than conventional synthesis. Second, a mixed pool may be generated and used for mutagenesis of multiple different genes. To use the same oligo-pool for mutagenesis of a variety of genes, the user must only quantify the fraction of the oligo-pool specific to her mutagenic experiment and adjust the volume and effective concentration of the oligo-pool for use in nicking mutagenesis.

User-defined single pot mutagenesis using unamplified oligo pools.

User-defined mutagenic libraries are fundamental for applied protein engineering workflows. Here we show that unamplified oligo pools can be used to prepare site saturation mutagenesis libraries from plasmid DNA with near-complete coverage of desired mutations and few off-target mutations. We find that oligo pools yield higher quality libraries when compared to individually synthesized degenerate oligos. We also show that multiple libraries can be multiplexed into a single oligo pool, making preparation of multiple libraries less expensive and more convenient. We provide software for automatic oligo pool design that can generate mutagenic oligos for saturating or focused libraries.

Characterizing Protein-Protein Interactions Using Deep Sequencing Coupled to Yeast Surface Display.

In this chapter, we discuss a method to determine the affinity and specificity of nearly all single-point mutants for a full-length protein binder. This method combines deep sequencing, comprehensive mutagenesis, yeast surface display, and fluorescence-activated cell sorting. This approach has been used to study sequence-function relationships for protein-protein interactions. The data can be used to determine the fine conformational epitope on the protein binder.