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Related Concept Videos

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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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Homologous Recombination02:31

Homologous Recombination

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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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CRISPR and crRNAs02:53

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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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One of the common DNA damages is the chemical alteration of single bases by alkylation, oxidation, or deamination. The altered bases cause mispairing and strand breakage during replication. This type of damage causes minimal change to the DNA double helix structure and can be repaired by the base excision repair (BER) pathways. BER corrects damaged DNA sequences by removing the damaged base and restoring the original base sequence using the complementary strand as a template.
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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/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art
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CRISPR Nickase-Mediated Base Editing in Yeast.

Kouichi Kuroda1, Mitsuyoshi Ueda2

  • 1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.

Methods in Molecular Biology (Clifton, N.J.)
|September 5, 2020
PubMed
Summary
This summary is machine-generated.

A new CRISPR nickase system allows precise, whole-genome base editing in yeast. This method avoids double-strand breaks, reducing off-target mutations and enabling faster, more accurate genome editing.

Keywords:
CAN1CRISPR/Cas9Gap repair cloningGenome editingNickaseSaccharomyces cerevisiae

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is a powerful tool for genome editing.
  • Existing CRISPR/Cas9 systems face limitations including off-target effects and restricted editing precision to specific DNA sequences.
  • Precise editing is often confined to the 20-base pair guide RNA (gRNA) targeting site and the protospacer adjacent motif (PAM) sequence.

Purpose of the Study:

  • To develop a novel CRISPR nickase system for precise, genome-wide base editing in Saccharomyces cerevisiae.
  • To overcome the limitations of traditional CRISPR/Cas9 systems regarding off-target effects and editing scope.
  • To establish a convenient and efficient method for precise genome editing in yeast.

Main Methods:

  • Development of a CRISPR nickase system utilizing a single Cas9 nickase.
  • Application of the system in Saccharomyces cerevisiae for base editing.
  • Integration with yeast gap repair cloning for streamlined genome editing.
  • Assessment of off-target mutations and editing precision.

Main Results:

  • The developed CRISPR nickase system enables precise genome-wide base editing.
  • Avoidance of double-strand breaks (DSB) and subsequent non-homologous end joining (NHEJ) leads to broader precise editing.
  • No unintended mutations were detected at off-target sites.
  • Precise genome editing in yeast cells was achieved within 5 days.

Conclusions:

  • The novel CRISPR nickase system offers a significant advancement for precise and convenient genome editing in yeast.
  • This method enhances editing accuracy and expands the editable genomic region compared to conventional CRISPR/Cas9.
  • The system's efficiency and lack of off-target mutations make it a valuable tool for genetic research and engineering in yeast.