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Related Concept Videos

In-vitro Mutagenesis01:16

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To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
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Mutations in Microorganisms01:18

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Mutations are heritable changes in an organism’s genome involving alterations in the base sequence of DNA or RNA. These changes can influence cellular processes and phenotypic traits, potentially transforming the unaltered wild type into a mutant form. Such changes, termed forward mutations, are pivotal in shaping the genetic diversity of organisms.RNA viruses exhibit the highest mutation rates due to the absence of robust proofreading mechanisms during genome replication. In contrast,...
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Conservative Site-specific Recombination and Phase Variation02:53

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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
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Updated: Feb 20, 2026

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
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Targeted mutagenesis: A sniper-like diversity generator in microbial engineering.

Xiang Zheng1, Xin-Hui Xing1, Chong Zhang1

  • 1Key Laboratory for Industrial Biocatalysis, Ministry of Education, Institute of Biochemical Engineering, Department of Chemical Engineering, Center for Synthetic & Systems Biology, Tsinghua University, Beijing 100084, China.

Synthetic and Systems Biotechnology
|October 25, 2017
PubMed
Summary

Mutations drive evolution and are key to engineering molecules and microbes. This review classifies targeted mutagenesis techniques into in vitro and in vivo methods, exploring their mechanisms and applications for future evolution research.

Keywords:
3′-LTR, 3’-long terminal repeat5-FOA, 5-fluoro-orotic acidCRISPR/Cas9, clustered regularly interspaced short palindromic repeats and associated protein 9DNA Pol III, DNA polymerase IIIDNA PolI, DNA polymerase IDSB, double strand breakEvolutionFLASH, fast ligation-based automatable solid-phase high-throughputHDR, homology-directed repairHIV, human immunodeficiency virusICE, in vivo continuous evolutionLIC, ligation-independent cloningMAGE, multiplex automated genome engineeringMMEJ, microhomology-mediated end-joiningMutationsNHEJ, error-prone non-homologous end-joiningORF, open reading framePAM, protospacer-adjacent motifRVD, repeat variable di-residueSynthetic biologyTALE, transcription activator-like effectorTALEN, transcription activator-like effector nucleaseTP, terminal proteinTP-DNAP, TP-DNA polymerase fusionTaGTEAM, targeting glycosylase to embedded arrays for mutagenesisTargeted mutagenesisYOGE, yeast oligo-mediated genome engineeringZF, zinc-finger proteinZFN, zinc-finger nucleasedCas9, catalytically dead Cas9dNTP, deoxy-ribonucleoside triphosphatedsDNA, double-stranded DNAerror-prone PCR, error-prone polymerase chain reactionnon-GMO, non-genetically modified organismpre-crRNA, pre-CRISPR RNAsctetR, single chain tetRsgRNA, single-guide RNAssDNA, single-stranded DNAtracrRNA, trans-encoded RNA

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Area of Science:

  • Molecular Biology
  • Evolutionary Biology
  • Biotechnology

Background:

  • Mutations are fundamental to evolution and are widely used for engineering biological functions.
  • Global and targeted mutagenesis are primary methods for generating mutations, differing in their scope.
  • Targeted mutagenesis offers controlled manipulation, preventing evolutionary escape and error catastrophe.

Purpose of the Study:

  • To review and classify existing targeted mutagenesis techniques.
  • To differentiate methods based on in vitro and in vivo diversity generation.
  • To discuss mechanisms, applications, and future trends in targeted mutagenesis.

Main Methods:

  • Classification of targeted mutagenesis techniques into in vitro and in vivo categories.
  • Review of mechanisms and applications for each category.
  • Analysis of interconnections and future development trends.

Main Results:

  • Targeted mutagenesis techniques are categorized based on in vitro or in vivo diversity generation.
  • Mechanisms and applications of both in vitro and in vivo methods are detailed.
  • Insights into the connections and future directions of these techniques are provided.

Conclusions:

  • Targeted mutagenesis is crucial for controlled evolution and functional engineering.
  • The in vitro and in vivo classifications provide a framework for understanding current techniques.
  • Future research trends point towards advancements in next-generation evolution strategies.