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

Abstract

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.

An integrated technology for quantitative wide mutational scanning of human antibody Fab libraries

“…To demonstrate the capability of MAGMA-seq to track potential development trajectories of multiple antibodies simultaneously, we selected three anti-S1 antibodies [29][30][31] that target Wuhan Hu-1 S1 at two distinct domains, the RBD and the NTD (Figure 2a). For each of these antibodies, mutagenic libraries theoretically comprising all possible sets of mutation between the mature and inferred universal common ancestor (UCA) were constructed using combinatorial nicking mutagenesis 32,33 and the libraries were pooled in approximately equimolar ratios and assembled into the yeast surface vector with a target of multiple barcodes per antibody variant (Figure 2a).…”

SUNi mutagenesis: scalable and uniform nicking for efficient generation of variant libraries

“…1). Previous data indicated that longer homology arms could improve mutagenesis efficiency [17], and that the melting temperature (T m ) of the mutagenic primer was correlated with mutagenesis efficiency [18]. We reasoned that since binding of both homology arms to the template is required for efficient mutagenesis, performance could be improved by optimizing the T m of both arms of the primer independently.…”

Oligo Pools as an Affordable Source of Synthetic DNA for Cost‐Effective Library Construction in Protein‐ and Metabolic Pathway Engineering

“…After the n/a: not available; [a] The average of the two reported independent runs of optimized multi-site NM is given (Table 1 in Ref. [49]).…”

“…Double mutations were depleted in near adjacent positions, likely because a second oligonucleotide would either not anneal properly or overwrite the mutation from the first oligonucleotide. An optimized protocol for performing multi‐site NM has recently been developed [49] . This protocol uses model‐driven oligo design to achieve >99 % coverage of a multi‐site mutagenized 32.768 membered antibody library (Table 2).…”

“…An optimized protocol for performing multi‐site NM has recently been developed. [49] This protocol uses model‐driven oligo design to achieve >99 % coverage of a multi‐site mutagenized 32.768 membered antibody library (Table 2 ). The authors further provide an automated workflow for oligo pool design using a protein‐coding sequence as the sole input.…”