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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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CRISPR01:59

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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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Homologous Recombination02:31

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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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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.
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CRISPR and crRNAs02:53

CRISPR and crRNAs

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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
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Related Experiment Video

Updated: Sep 24, 2025

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins
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CRISPR-Cas Assisted Shotgun Mutagenesis Method for Evolutionary Genome Engineering.

Ming Zhao1,2, Miaomiao Gao1, Liangbin Xiong1,3

  • 1State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.

ACS Synthetic Biology
|May 2, 2022
PubMed
Summary

We developed CRISPR-Cas assisted random mutation (CARM) for whole-genome mutagenesis. This technique rapidly enhances organism traits, demonstrated by a 10.5-fold increase in yeast beta-carotene production.

Keywords:
CRISPR-mediated genome engineeringiterative evolutionwhole-genome mutagenesis

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Area of Science:

  • Molecular Biology
  • Genetics
  • Synthetic Biology

Background:

  • Genome mutagenesis is a key driver of organismal evolution.
  • Targeted genome editing tools like CRISPR-Cas offer precise modifications but often lack capacity for broad genetic diversity generation.

Purpose of the Study:

  • To develop a novel whole-genome mutagenesis technique for rapid generation of genetic diversity.
  • To demonstrate the utility of this technique for evolving specific metabolic traits in yeast.

Main Methods:

  • Developed CRISPR-Cas assisted random mutation (CARM) using a random gRNA library and SpCas9-NG for shotgun-like genome damage.
  • Applied CARM to evolve beta-carotene production in *Saccharomyces cerevisiae* BY4741 over seven rounds.
  • Utilized CARM for S-adenosyl-L-methionine production enhancement in *S. cerevisiae* CEN.PK2-1C.

Main Results:

  • Isolated a beta-carotene hyperproducing yeast strain (C7-143) with a 10.5-fold increase in production and 857 diverse genomic mutations (indels, duplications, inversions, rearrangements).
  • Observed significant alterations in 2541 gene expressions in the evolved strain, indicating deep metabolic reconstruction.
  • Achieved a 2.28-fold increase in S-adenosyl-L-methionine production in *S. cerevisiae* CEN.PK2-1C after one CARM round.

Conclusions:

  • CARM is an effective technique for generating extensive genomic diversity and rapidly evolving desired phenotypes.
  • The method enables deep metabolic reconstruction and can be applied to various industrial yeast strains.
  • CARM facilitates the identification of novel phenotypes for further investigation and reverse engineering.