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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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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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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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CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art
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Improved genome editing by an engineered CRISPR-Cas12a.

Enbo Ma1,2, Kai Chen1,2, Honglue Shi1,3

  • 1Innovative Genomics Institute, University of California, Berkeley, CA 94720, USA.

Nucleic Acids Research
|December 20, 2022
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Summary

CRISPR-Cas12a genome editing was improved by engineering enzyme activity. Mutants showed enhanced homology-directed repair (HDR) and editing efficiency, creating better genome editing tools.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • CRISPR-Cas12a is a programmable genome editing enzyme.
  • Unlike Cas9, Cas12a has cis- and trans-DNase activities.
  • Understanding the balance of these activities is key for genome editing.

Purpose of the Study:

  • To investigate how cis- vs. trans-DNase activity affects Cas12a genome editing.
  • To engineer Cas12a variants with improved genome editing efficiency.
  • To develop a pipeline for creating enhanced CRISPR-Cas genome editing tools.

Main Methods:

  • Structure-guided engineering of Lachnospiraceae bacterium Cas12a to disrupt trans-activity.
  • Directed evolution to generate additional Cas12a mutants.
  • Assessing in vitro DNase activity and in human cell genome editing.

Main Results:

  • Engineered Cas12a mutants with disrupted trans-activity showed minimal genome editing.
  • Directed evolution yielded mutants with robust genome editing and enhanced homology-directed repair (HDR) by 2-18 fold.
  • Improved Cas12a (iCas12a) variants demonstrated superior editing efficiency at difficult genomic sites.

Conclusions:

  • Selective disruption of Cas12a trans-activity is crucial for efficient genome editing.
  • Combining structural insights with directed evolution creates a powerful strategy for improving CRISPR-Cas tools.
  • This approach can be applied to enhance other genome editing proteins.