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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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CRISPR01:59

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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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Conservative Site-specific Recombination and Phase Variation02:53

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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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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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Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9
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Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production.

Colby J Feser1, Christopher J Lees1, Daniel T Lammers2

  • 1Department of Pediatrics, Division of Blood and Marrow Transplantation, MMC 366 Mayo, 8366A, 420 Delaware Street SE, Minneapolis, MN 55455, USA.

International Journal of Molecular Sciences
|May 14, 2022
PubMed
Summary
This summary is machine-generated.

This study introduces a novel CRISPR-Cas9 Synergistic Activation Mediator (SAM) system for efficient production of multiple hemostatic agents in human cells, improving upon current recombinant methods.

Keywords:
CRISPRcoagulationfibrinogenmultiplexingrecombinant protein

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Genetic Engineering

Background:

  • Current hemostatic agents rely on pooled plasma, necessitating costly donor screening and processing.
  • Existing recombinant production methods often focus on single gene products and utilize non-human cells.

Purpose of the Study:

  • To develop an improved recombinant production system for hemostatic agents using human cells.
  • To demonstrate the capability of CRISPR-Cas9 Synergistic Activation Mediators (SAMs) for multiplex gene product generation.

Main Methods:

  • Engineered CRISPR-Cas9 SAMs for targeted overexpression of coagulation factors II, VII, IX, X, and fibrinogen in Human Embryonic Kidney (HEK293) cells.
  • Assessed gene upregulation using quantitative RT-PCR (qRT-PCR) and protein expression via ELISA analysis for both singleplex and multiplex systems.

Main Results:

  • Achieved significant gene expression increases: 120-4700-fold for coagulation factors (singleplex) and 60-680-fold (multiplex).
  • Demonstrated substantial fibrinogen subunit gene activation: 1700-92,000-fold (singleplex) and 80-5500-fold (multiplex).
  • Confirmed concomitant protein upregulation through ELISA analysis.

Conclusions:

  • CRISPR-Cas9 SAMs enable efficient single and multi-agent production in human cells.
  • This approach represents a significant engineering advancement for recombinant peptide production, enhancing supply, uniformity, and safety.