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Related Concept Videos

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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Genome Editing in Mammalian Cell Lines using CRISPR-Cas
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Operating CRISPR/Cas12a in a complex nucleic acid sequence background.

Henning Hellmer1, Thomas Mayer1, Lea Bauersachs1

  • 1Department of Bioscience, School of Natural Sciences, Technical University of Munich, Garching, D-85748, Germany.

Nucleic Acids Research
|April 30, 2026
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Summary
This summary is machine-generated.

CRISPR-Cas12a activity in complex environments is hindered by background nucleic acids. Guide RNA seed region properties, like GC content, influence this interference, impacting biosensing and gene editing applications.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • CRISPR-Cas systems, including Cas12a, are vital tools for genome editing and biosensing due to their RNA-guided specificity.
  • Performance optimization in biological settings often focuses on guide RNA (gRNA) design and minimizing off-target effects.
  • Interference from background nucleic acids can kinetically inhibit CRISPR-Cas12a interactions, particularly in complex biological samples.

Purpose of the Study:

  • To investigate the kinetic inhibition of Cas12a by non-cognate background nucleic acids.
  • To determine how background single-stranded RNA and double-stranded DNA affect CRISPR-Cas12a reaction kinetics.
  • To identify guide RNA (gRNA) sequence features that mitigate this inhibitory effect.

Main Methods:

  • In vitro kinetic assays were performed under controlled conditions.
  • The influence of background single-stranded RNA and double-stranded DNA on Cas12a activity was systematically evaluated.
  • Experiments utilized varying gRNA seed region compositions, including GC content and purine-to-pyrimidine ratios.
  • Cell-based gene activation assays with deactivated Cas12a (dCas12a) were conducted to assess in vivo relevance.

Main Results:

  • Background nucleic acids significantly inhibit the kinetic interaction between Cas12a and its target.
  • The purine-to-pyrimidine ratio and GC content within the gRNA seed region critically influence the degree of kinetic inhibition.
  • gRNAs with low GC content and a high purine fraction in the seed region showed reduced susceptibility to background interference.
  • A gRNA with high uracil content in the seed region demonstrated pronounced inhibition when exposed to double-stranded DNA.

Conclusions:

  • Guide RNA seed region composition is a key factor in overcoming kinetic inhibition by background nucleic acids in CRISPR-Cas12a systems.
  • Findings suggest strategies for designing more robust gRNAs for CRISPR-Cas12a applications in complex biological matrices.
  • The in vitro results have implications for optimizing CRISPR-Cas12a-based biosensing and gene editing tools for in vivo use.