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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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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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Assessing Temperature-Dependent DNA Cleavage by CRISPR-Cas9.

Alexa L Knight1, Jinping Luo2, George P Lisi1,3

  • 1Department of Molecular Biology, Cell Biology & Biochemistry, Brown University, Providence, RI, USA.

Bio-Protocol
|October 13, 2025
PubMed
Summary
This summary is machine-generated.

This study presents a new protocol for measuring CRISPR-Cas9 DNA cleavage activity across various temperatures. This method helps in understanding and engineering Cas9 enzymes for diverse applications, including therapeutic uses.

Keywords:
CRISPR-Cas9DNA cleavageEndonucleaseEnzymeThermophile

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biophysics

Background:

  • CRISPR-Cas9 is a powerful gene-editing tool with therapeutic potential, relying on DNA cleavage guided by RNA and PAM recognition.
  • Cas9 activation involves an allosteric mechanism, influencing its cleavage activity and specificity.
  • Biophysical studies aim to engineer enhanced Cas effectors by understanding allosteric effects.

Purpose of the Study:

  • To develop and validate a protocol for quantifying DNA cleavage by CRISPR-Cas9 systems across a range of temperatures.
  • To enable the assessment of temperature-dependent function in both mesophilic and thermophilic Cas9 orthologs.
  • To provide a versatile assay applicable to various Cas-RNP systems and genomic targets.

Main Methods:

  • Adaptation of established in vitro DNA cleavage assays to incorporate temperature-dependent measurements.
  • Utilizing Cas9 from thermophilic organisms (e.g., Geobacillus stearothermophilus) and mesophilic organisms (e.g., SpCas9).
  • Qualitative and quantitative assessment of DNA cleavage across different physiological temperature regimes.

Main Results:

  • The protocol successfully quantifies DNA cleavage activity for both mesophilic and thermophilic Cas9 systems.
  • It allows for the evaluation of functional temperature ranges, crucial for extremophilic Cas systems.
  • The assay is adaptable for different genomic loci and protospacer-adjacent motif (PAM) requirements.

Conclusions:

  • This validated protocol provides a robust method for assessing temperature-dependent DNA cleavage by CRISPR-Cas9.
  • It facilitates the engineering of enhanced Cas effectors by characterizing their thermal activity profiles.
  • The assay's adaptability makes it a valuable tool for diverse CRISPR-Cas9 research and development applications.