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Related Concept Videos

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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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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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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Orthogonal transcriptional modulation and gene editing using multiple CRISPR-Cas systems.

Amalie Dyrelund Broksø1, Louise Bendixen1, Simon Fammé1

  • 1Department of Biomedicine, Aarhus University, Aarhus C, Denmark.

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|November 20, 2024
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Summary

This study benchmarks RNA-delivered CRISPR gene editing tools for precise transcriptional control and gene knockout. Optimized CRISPR systems achieve efficient trimodal engineering in human T cells, paving the way for advanced regenerative medicine.

Keywords:
CRISPRCRISPRaCRISPRiCasactivationorthogonalorthologsrepressiontranscriptionaltrimodal

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Synthetic Biology

Background:

  • CRISPR-Cas systems, including CRISPRa and CRISPRi, offer programmable gene regulation via dCas fusion or recruitment.
  • RNA delivery of CRISPR components is an emerging strategy for transient gene modulation.
  • Orthogonal CRISPR systems enable simultaneous gene upregulation and downregulation.

Purpose of the Study:

  • To benchmark combinations of RNA-delivered dCas and transcriptional modulators for gene regulation.
  • To establish trimodal genetic engineering by combining CRISPRa/CRISPRi with CRISPR-mediated gene knockout.
  • To optimize parameters for simplified trimodal engineering using truncated sgRNAs and SpCas9.

Main Methods:

  • Utilized Staphylococcus aureus and Streptococcus pyogenes dCas9 for orthogonal transcriptional modulation.
  • Implemented Cas12a ribonucleoprotein delivery for gene knockout.
  • Investigated the impact of Cas9 protein/sgRNA ratio on knockout and activation efficiencies.
  • Performed trimodal genetic engineering in primary human T cells.

Main Results:

  • Demonstrated efficient orthogonal transcriptional modulation (upregulation and downregulation) using RNA-delivered dCas9.
  • Successfully established trimodal genetic engineering combining transcriptional regulation and gene knockout.
  • Identified the Cas9 protein/sgRNA ratio as critical for balancing gene editing outcomes.
  • Achieved high trimodal engineering efficiencies in primary human T cells with preserved cell health.

Conclusions:

  • RNA-delivered CRISPR systems provide versatile tools for complex genetic engineering.
  • Trimodal engineering enables simultaneous transient transcriptome modulation and permanent DNA changes in a single step.
  • This approach holds significant potential for applications in regenerative medicine and therapeutic development.