Related Experiment Video
Updated: Jul 26, 2025
11:53
Substrate Generation for Endonucleases of CRISPR/Cas Systems
Published on: September 8, 2012
27.4K
[Type II CRISPR-Cas System Nucleases: A Pipeline for Prediction and In Vitro Characterization]
A A Vasileva1,2,3, S A Aliukas4, P A Selkova1,2
1Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, 123182 Russia.
Molekuliarnaia Biologiia
|June 16, 2023
Summary
Researchers developed a workflow to discover and characterize novel Cas9 nucleases for genome editing. This method aids in overcoming limitations of current CRISPR-Cas9 tools for broader applications.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Context:
- CRISPR-Cas systems provide programmable genome editing capabilities.
- Cas9 nucleases are widely used for targeted DNA modifications.
- Existing Cas9 editors have limitations, necessitating the search for new variants.
Purpose:
- To present a workflow for identifying and characterizing novel Cas9 nucleases.
- To detail protocols for bioinformatical searching, protein isolation, and activity testing.
- To determine the PAM sequence requirements for new Cas9 variants.
Summary:
- This study outlines a comprehensive workflow for discovering and characterizing new Cas9 nucleases.
- Protocols cover bioinformatical screening, recombinant protein production, in vitro nuclease activity assays, and PAM sequence determination.
- The workflow addresses potential challenges and offers solutions for developing improved genome editing tools.
Impact:
- Facilitates the discovery of novel Cas9 variants with potentially enhanced or altered specificities.
- Contributes to the advancement of genome editing technologies for research and therapeutic applications.
- Provides a standardized methodology for the development and characterization of new CRISPR-Cas9 tools.
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