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CRISPR

CRISPR

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...

CRISPR and crRNAs

CRISPR and crRNAs

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.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...

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Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics

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PAM-Engineered Toehold Switches as Input-Responsive Activators of CRISPR-Cas12a for Sensing Applications.

Neda Bagheri¹, Alejandro Chamorro¹, Andrea Idili¹

¹Department of Sciences and Chemical Technologies, University of Rome, Tor Vergata, Via della Ricerca Scientifica 1, 00133, Rome, Italy.

Angewandte Chemie (International Ed. in English)

|January 29, 2024

Summary

Researchers engineered CRISPR-Cas12a systems using Toehold Switch DNA hairpins to control gene editing and diagnostics. This innovation enables precise molecular input-triggered Cas12a activation for advanced biotechnological applications.

Keywords:

CRISPR biosensors Cas12a PAM strand displacement reaction trans-cleavage

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Area of Science:

Biotechnology
Molecular Biology
CRISPR Technology

Background:

CRISPR-Cas12a is a versatile tool for gene editing and diagnostics.
Controlling Cas12a activity requires advanced bio-engineering strategies.

Purpose of the Study:

To develop a novel method for controlling Cas12a activity using engineered DNA structures.
To demonstrate the application of this system in molecular sensing and diagnostics.

Main Methods:

Engineered Toehold Switch DNA hairpins with a locked protospacer adjacent motif (PAM).
Utilized strand displacement reactions to reconfigure DNA conformation and modulate PAM accessibility.
Leveraged Cas12a's trans-cleavage activity for signal transduction.

Main Results:

Demonstrated successful control over Cas12a binding and cleavage activities.
Showcased proximity-based strand displacement to trigger Cas12a activity upon target binding.
Achieved rapid, one-pot detection of IgG antibodies and small molecules with high sensitivity and specificity.

Conclusions:

Engineered Toehold Switches provide a programmable platform for regulating Cas12a.
This approach offers versatile sensing capabilities for various analytes in complex matrices.
The system holds significant promise for diverse biotechnological and bioanalytical applications.