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Updated: May 18, 2026

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Published on: September 8, 2012

Substrate generation for endonucleases of CRISPR/cas systems.

Judith Zoephel¹, Srivatsa Dwarakanath, Hagen Richter

¹Prokaryotic Small RNA Biology, Max-Planck-Institute for Terrestrial Microbiology.

Journal of Visualized Experiments : Jove

|September 19, 2012

Summary

Prokaryotes use CRISPR/Cas systems as adaptive immunity against viruses. This study presents methods to generate RNA molecules for studying Cas endoribonucleases, crucial for understanding this defense mechanism.

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Area of Science:

Microbiology
Molecular Biology
Genetics

Background:

Prokaryotic evolution is shaped by virus-host interactions.
Prokaryotes evolved defense mechanisms like CRISPR/Cas systems against viral infections.
CRISPR/Cas systems are adaptive immune systems composed of CRISPR sequences and Cas genes.

Purpose of the Study:

To present methods for generating crRNAs and precursor-cRNAs.
To facilitate the study of Cas endoribonucleases.
To investigate the function and diversity of Cas proteins.

Main Methods:

In vitro T7 RNA polymerase run-off transcription for generating longer RNA sequences.
Synthesis of short RNA oligonucleotides.
Incorporation of radioactive nucleotides for labeled substrates.
Generation of synthetic or mutant crRNAs.

Main Results:

Methods for producing crRNAs and precursor-cRNAs are established.
These methods allow for the creation of labeled and modified RNA substrates.
Cas6 endonuclease activity is used to process precursor RNAs into mature crRNAs.

Conclusions:

The presented methods enable detailed study of Cas endoribonucleases.
Understanding Cas proteins is key to elucidating CRISPR/Cas system diversity and function.
This research contributes to the study of prokaryotic adaptive immunity.