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CRISPR/Cas9 Genome Editing01:28

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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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High-performance CRISPR-Cas12a genome editing for combinatorial genetic screening.

Rodrigo A Gier1,2,3,4, Krista A Budinich1,2,3, Niklaus H Evitt1,2,3,5

  • 1Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.

Nature Communications
|July 15, 2020
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Summary

This study introduces an optimized CRISPR-Cas12a system for combinatorial genetic screening in cancer cells. The novel approach efficiently identifies gene target synergies, revealing new synthetic sick interactions in leukemia.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cancer Research

Background:

  • CRISPR-based genetic screening is vital for cancer drug target discovery.
  • Multiplex gene editing for revealing gene target synergies presents a significant challenge.

Purpose of the Study:

  • To develop a robust CRISPR-Cas12a-based method for combinatorial genetic screening in cancer cells.
  • To enable high-efficiency screening for gene target interactions and drug discovery.

Main Methods:

  • Engineering the CRISPR-AsCas12a system with modifications to the Cas protein and CRISPR RNA (crRNA).
  • Implementing an optimized AsCas12a (opAsCas12a) for double knockout screening.
  • Screening against epigenetic regulators in leukemia cells.

Main Results:

  • Demonstrated high efficiency of the opAsCas12a system for combinatorial screening.
  • Identified synthetic sick interactions between specific gene pairs: Brd9 & JMJD6, KAT6A & JMJD6, and BRPF1 & JMJD6.
  • Validated the system's performance in uncovering gene target synergies.

Conclusions:

  • The developed CRISPR-AsCas12a approach offers a simple and robust solution for combinatorial genetic screening.
  • This method facilitates the discovery of synergistic gene interactions crucial for cancer therapeutic strategies.
  • The findings provide novel insights into epigenetic regulator interactions in leukemia.