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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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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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Yeast Signaling01:28

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Yeasts are single-celled organisms, but unlike bacteria, they are eukaryotes (cells with a nucleus). Cell signaling in yeast is similar to signaling in other eukaryotic cells. A ligand, such as a protein or a small molecule released from a yeast cell, attaches to a receptor on the cell surface. The binding stimulates second-messenger kinases to activate or inactivate transcription factors that further regulate gene expression. Many of the yeast intracellular signaling cascades have similar...
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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/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation
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Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9.

Azat Akhmetov1,2, Jon M Laurent1,3, Jimmy Gollihar1

  • 1Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.

Bio-Protocol
|May 18, 2018
PubMed
Summary

This study introduces a single-step CRISPR-Cas9 genome editing method for budding yeast. This efficient technique modifies essential and nonessential genes without selectable markers, simplifying yeast genetic research.

Keywords:
CRISPRGenome editingHomologous recombinationHumanizationOrtholog complementationYeast engineering

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Traditional yeast genome modification often requires multi-step procedures involving selectable markers.
  • Existing methods can be inefficient or complex for targeting essential genes in budding yeast.
  • Homology-directed repair is crucial for yeast genome engineering but requires optimized strategies.

Purpose of the Study:

  • To develop a streamlined, single-step CRISPR-Cas9 genome editing protocol for budding yeast.
  • To enable modification of both essential and nonessential genes without the need for selectable markers.
  • To demonstrate the application of this method for gene replacement, including essential genes with human orthologs.

Main Methods:

  • Utilized CRISPR-Cas9 nuclease to create targeted double-stranded DNA breaks in the yeast genome.
  • Leveraged the cell's inherent, but often inefficient, non-homologous end-joining repair pathway to induce lethality.
  • Employed PCR-generated repair templates to facilitate efficient homology-directed repair for precise gene editing.

Main Results:

  • Achieved single-step genome modification in budding yeast, bypassing the need for selectable markers.
  • Demonstrated successful editing of essential genes, where the requirement for a functional allele acts as a selection.
  • Successfully replaced the essential yeast gene HEM2 with its human ortholog ALAD as a proof-of-concept.

Conclusions:

  • The described CRISPR-Cas9 method provides a highly efficient and simplified approach for yeast genome editing.
  • This protocol is versatile, applicable to essential and nonessential genes, and reduces experimental complexity.
  • The strategy facilitates functional studies by enabling precise gene replacement, including interspecies gene swapping.