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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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Homologous Recombination02:31

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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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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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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

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Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system.

Kabin Xie1, Bastian Minkenberg1, Yinong Yang2

  • 1Department of Plant Pathology and Environmental Microbiology and the Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA 16802.

Proceedings of the National Academy of Sciences of the United States of America
|March 4, 2015
PubMed
Summary
This summary is machine-generated.

Researchers engineered a tRNA-processing system to create multiple guide RNAs (gRNAs) from a single gene, enhancing the CRISPR/Cas9 genome editing tool for multiplex editing and crop improvement.

Keywords:
CRISPR/Cas9genome editingmultiplextRNA processing

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is a key technology for genome engineering.
  • The targeting specificity and multiplexing capability of CRISPR/Cas9 are limited by the efficiency of guide RNA (gRNA) expression systems.

Purpose of the Study:

  • To develop a novel strategy for producing multiple gRNAs from a single polycistronic gene.
  • To enhance the targeting and multiplex genome editing efficiency of the CRISPR/Cas9 system.

Main Methods:

  • Engineered the endogenous tRNA-processing system to process synthetic genes containing tandemly arrayed tRNA-gRNA architecture.
  • Demonstrated in vivo processing of tRNA-gRNA constructs into functional gRNAs with specific 5' targeting sequences.
  • Applied the method for multiplex genome editing and chromosomal-fragment deletion in transgenic rice plants.

Main Results:

  • Achieved efficient and precise processing of synthetic tRNA-gRNA genes into functional gRNAs.
  • Successfully directed Cas9 to edit multiple chromosomal targets simultaneously.
  • Demonstrated high efficiency (up to 100%) for multiplex genome editing and chromosomal-fragment deletion in rice.

Conclusions:

  • The engineered tRNA-processing system provides a robust platform for boosting CRISPR/Cas9 targeting and multiplex editing capabilities.
  • This method is broadly applicable across organisms due to the conservation of tRNA and its processing machinery.
  • The strategy offers a significant advancement for genome engineering applications in research and crop improvement.