title: Codon compression algorithms for saturation mutagenesis.
abstract: Saturation mutagenesis is employed in protein engineering and genome-editing efforts to generate libraries that span amino acid design space. Traditionally, this is accomplished by using degenerate/compressed codons such as NNK (N = A/C/G/T, K = G/T), which covers all amino acids and one stop codon. These solutions suffer from two types of redundancy: (a) different codons for the same amino acid lead to bias, and (b) wild type amino acid is included within the library. These redundancies increase library size and downstream screening efforts. Here, we present a dynamic approach to compress codons for any desired list of amino acids, taking into account codon usage. This results in a unique codon collection for every amino acid to be mutated, with the desired redundancy level. Finally, we demonstrate that this approach can be used to design precise oligo libraries amendable to recombineering and CRISPR-based genome editing to obtain a diverse population with high efficiency.
authors.affiliation: †Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado 80309, United States.
authors.forename: Gur
authors.initials: G
authors.lastname: Pines
authors.forename: Assaf
authors.initials: A
authors.lastname: Pines
authors.affiliation: †Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado 80309, United States.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Garst
authors.affiliation: †Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado 80309, United States.
authors.forename: Ramsey I
authors.initials: RI
authors.lastname: Zeitoun
authors.affiliation: †Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado 80309, United States.|‡Biosciences Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, Colorado 80401, United States.
authors.forename: Sean A
authors.initials: SA
authors.lastname: Lynch
authors.affiliation: †Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado 80309, United States.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Gill
mesh_terms: D000465:Algorithms; D000596:Amino Acids / Q000235:genetics; D003062:Codon / Q000235:genetics*; D015723:Gene Library; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics; D015202:Protein Engineering / Q000379:methods
chemical_list: D000596:Amino Acids; D003062:Codon; D009841:Oligonucleotides
keywords: CRISPR selection; codon redundancy; codon usage; genome editing; library size; saturation mutagenesis