title: Polishing the craft of genetic diversity creation in directed evolution.
abstract: Genetic diversity creation is a core technology in directed evolution where a high quality mutant library is crucial to its success. Owing to its importance, the technology in genetic diversity creation has seen rapid development over the years and its application has diversified into other fields of scientific research. The advances in molecular cloning and mutagenesis since 2008 were reviewed. Specifically, new cloning techniques were classified based on their principles of complementary overhangs, homologous sequences, overlapping PCR and megaprimers and the advantages, drawbacks and performances of these methods were highlighted. New mutagenesis methods developed for random mutagenesis, focused mutagenesis and DNA recombination were surveyed. The technical requirements of these methods and the mutational spectra were compared and discussed with references to commonly used techniques. The trends of mutant library preparation were summarised. Challenges in genetic diversity creation were discussed with emphases on creating "smart" libraries, controlling the mutagenesis spectrum and specific challenges in each group of mutagenesis methods. An outline of the wider applications of genetic diversity creation includes genome engineering, viral evolution, metagenomics and a study of protein functions. The review ends with an outlook for genetic diversity creation and the prospective developments that can have future impact in this field.
authors.affiliation: Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, England, United Kingdom.
authors.forename: Kang Lan
authors.initials: KL
authors.lastname: Tee
authors.forename: Tuck Seng
authors.initials: TS
authors.lastname: Wong
mesh_terms: D000818:Animals; D057005:Bioengineering; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D015723:Gene Library; D014644:Genetic Variation; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed
keywords: 2′-Deoxy-P-nucleoside-5′-triphosphate; 2′-Deoxyinosine 5′-triphosphate; 7-Deaza-7-(triethylsilylethynyl)deoxyadenosine triphosphate; 8-Oxo-2′-deoxyguanosine-5′-triphosphate; 8-oxo-dGTP; AAV; ABI-REC; Adeno-associated virus; Asymmetric Bridge PCR with Intramolecular Homologous Recombination; Base pairs; CAST; CPEC; CRP; Circular Polymerase Extension Cloning; Combinatorial Active-Site Saturation Test; DGRs; DNA; DNA recombination; Deoxyribonucleic acid; Deoxyribonucleotide triphosphate; Directed evolution; Diversity-generating retroelements; Double-stranded DNA; DuARCheM; Dual Approach to Random Chemical Mutagenesis; EMP; EMS; Enoyl-acyl carrier protein reductase; Error-prone polymerase chain reaction; Error-prone rolling circle amplification; Ethyl methane sulfonate; Exponential Megapriming PCR; GOI; GST; Gene of interest; Genetic diversity; Genome engineering; Glutathione-S-transferase; ITCHY; InDel; Incremental Truncation for the Creation of Hybrid Enzymes; Insertion and deletion; KF; Klenow fragment; MAP; MEGAWHOP; MGS; MLF-SDM; Megaprimed and Ligase-Free PCR-based Method for Site-Directed Mutagenesis; Megaprimer PCR of Whole Plasmid; Metabolic engineering; Mutagenesis Assistant Program; Mutation Generation System; NEB; NRR; New England Biolabs; NiDE; Nicking DNA Endonuclease; Non-homologous random recombination; Nucleotide; OLTA; OSCARR; One-pot Simple Methodology for Casette Randomization and Recombination; OverLap extension PCR and TA cloning; PCR; PCR Production of Circular Plasmid; PERMUTE; PERMutation Using Transposase Engineering; PFLF-MSDM; PLICing; PPCP; PS; PTRec; Phosphorothioate; Phosphorothioate-based DNA Recombination; Phosphorothioate-based Ligase-Independent Gene Cloning; Phosphorylation-Free and Ligase-Free PCR-based Method for Multiple SDM; Polymerase chain reaction; RCA; REs; RF cloning; RGEN; RNA-guided Endonuclease; Random mutagenesis; Restriction enzymes; Restriction-Free cloning; Rolling circle amplification; SDM; SEFC; SHIPREC; SLiCE; SPRINP; STRU-Cloning; SeSaM; Seamless Enzyme-Free Cloning; Seamless Ligation Cloning Extract; Sequence Homology-Independent Protein RECombination; Sequence Saturation Mutagenesis; Single-Primer Reactions In Parallel; Single-Tube Restriction-based Ultrafiltration Cloning; Single-stranded DNA; Site-directed mutagenesis; StEP; Staggered Extension Process; Synthetic biology; T(s); T(v); TALENs; TAM; TIM; TMGS-PCR; TPCR; TRINS; TaGTEAM; Tandem Repeat Insertion; Targeting Glycosylases To Embedded Arrays for Mutagenesis; Transcription activator-like effector nucleases; Transcription-associated mutation; Transfer-PCR; Transitions; Transposon Integration mediated Mutagenesis; Transversions; TriNEx; TriNucleotide EXchange; Truncated Metagenomic Gene-Specific PCR; UDG; USER Friendly DNA Recombination; USERec; Uracil-DNA glycosylase; ZFN; Zinc finger nuclease; bp; cAMP receptor protein; dA(TESE)TP; dITP; dNTP; dPTP; dsDNA; enoyl ACP reductase; epPCR; epRCA; nt; p-Benzoylphenylalanine; pBpa; ssDNA