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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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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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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.
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Genomics is the science of genomes: it is the study of all the genetic material of an organism. In humans, the genome consists of information carried in 23 pairs of chromosomes in the nucleus, as well as mitochondrial DNA. In genomics, both coding and non-coding DNA is sequenced and analyzed. Genomics allows a better understanding of all living things, their evolution, and their diversity. It has a myriad of uses: for example, to build phylogenetic trees, to improve productivity and...
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RNA editing is a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the...
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CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art
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Efficient CRISPR-Cas9 mediated multiplex genome editing in yeasts.

Laiyou Wang1,2, Aihua Deng1, Yun Zhang1

  • 11CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101 China.

Biotechnology for Biofuels
|October 20, 2018
PubMed
Summary

A new CRISPR-Cas9-assisted multiplex genome editing (CMGE) method enables efficient genetic engineering in yeast. This tool facilitates multiplex gene knock-outs and multi-copy integrations, paving the way for enhanced biotechnological applications.

Keywords:
CRISPR–Cas9-assisted multiplex genome editingMarkerless multi-copy integrationMarkerless multi-locus integrationOgataea polymorphaSaccharomyces cerevisiae

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Area of Science:

  • Microbiology
  • Biotechnology
  • Synthetic Biology

Background:

  • The methylotrophic yeast *Ogataea polymorpha* is valuable for biotechnology but difficult to engineer due to limited genome editing tools.
  • Efficient genetic engineering is crucial for yeast cell factories in producing chemicals and biofuels.

Purpose of the Study:

  • To develop an efficient and versatile CRISPR-Cas9-assisted multiplex genome editing (CMGE) method for *Ogataea polymorpha*.
  • To establish methods for multiplex gene knock-outs and multi-locus/multi-copy integrations in yeast.

Main Methods:

  • Developed CRISPR-Cas9-assisted multiplex genome editing (CMGE) including multiplex gene knock-outs, multi-locus (ML), and multi-copy (MC) integration.
  • Applied CMGE for gene deletion, integration, and precise point mutation in *O. polymorpha*.
  • Utilized CMGE-ML for simultaneous integration of resveratrol biosynthesis genes and CMGE-MC for multi-copy gene integration.

Main Results:

  • Successfully achieved simultaneous integration of resveratrol biosynthesis pathway genes (*TAL*, *4CL*, *STS*) in *O. polymorpha* using CMGE-ML.
  • Demonstrated a ~20-fold increase in resveratrol production (97.23 mg/L) via multi-copy integration (CMGE-MC).
  • Achieved biosynthesis of human serum albumin and cadaverine in *O. polymorpha* using CMGE-MC, and validated CMGE-MC in *Saccharomyces cerevisiae*.

Conclusions:

  • An efficient and versatile multiplex genome editing method (CMGE) has been developed for yeast.
  • CMGE provides a powerful toolkit for genetic engineering and synthetic biology research in *O. polymorpha* and other yeast species.