ArticleProtein engineering, design & selection : PEDS2021
Optimization of multi-site nicking mutagenesis for generation of large, user-defined combinatorial libraries.
Article in Protein engineering, design & selection : PEDS, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed.
- Computational redesign of a thermostable T7 RNA polymerase.Protein engineering, design & selection : PEDS · 2026Article
- Computational redesign of a thermostable T7 RNA polymerase.bioRxiv : the preprint server for biology · 2025Article
- Retrospective SARS-CoV-2 human antibody development trajectories are largely sparse and permissive.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- GGAssembler: Precise and economical design and synthesis of combinatorial mutation libraries.Protein science : a publication of the Protein Society · 2024Article
- An integrated technology for quantitative wide mutational scanning of human antibody Fab libraries.Nature communications · 2024Article
- Positive epistasis drives clavulanic acid resistance in double mutant libraries of BlaC β-lactamase.Communications biology · 2024Article
- An integrated technology for quantitative wide mutational scanning of human antibody Fab libraries.bioRxiv : the preprint server for biology · 2024Article
- Protein engineering via sequence-performance mapping.Cell systems · 2023Review
- SUNi mutagenesis: Scalable and uniform nicking for efficient generation of variant libraries.PloS one · 2023Article
- Oligo Pools as an Affordable Source of Synthetic DNA for Cost-Effective Library Construction in Protein- and Metabolic Pathway Engineering.Chembiochem : a European journal of chemical biology · 2022Review
- Stabilization of the SARS-CoV-2 receptor binding domain by protein core redesign and deep mutational scanning.Protein engineering, design & selection : PEDS · 2022Article
- Stabilization of the SARS-CoV-2 Receptor Binding Domain by Protein Core Redesign and Deep Mutational Scanning.bioRxiv : the preprint server for biology · 2021Article
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Authors and funding
4 authors.
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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.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.