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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
Published on: June 16, 2017
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CRISPR-Cas12a exhibits metal-dependent specificity switching.
Giang T Nguyen1, Michael A Schelling1, Akshara Raju1
1Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA.
Nucleic Acids Research
|July 17, 2024
Summary
CRISPR-Cas12a gene editing tool specificity changes with magnesium ion concentration. Lowering Mg2+ enhances binding to some targets but increases errors from other mismatches, impacting its use in biotechnology.
Area of Science:
- Molecular Biology
- Biotechnology
- Genetics
Background:
- CRISPR-Cas12a is a key enzyme in CRISPR-Cas systems, widely used in genome editing.
- Previous specificity studies often used non-physiological magnesium concentrations.
- Understanding Cas12a specificity under cellular conditions is crucial for its applications.
Purpose of the Study:
- To investigate how magnesium ion (Mg2+) concentration affects Cas12a specificity.
- To compare the Mg2+-dependent specificity switching of different Cas12a orthologs.
- To understand the implications for CRISPR-Cas12a applications and natural immunity.
Main Methods:
- Profiling Cas12a ortholog specificity across a range of Mg2+ concentrations in vitro.
- Analyzing cleavage efficiency with varying seed and PAM-distal mismatches.
- Comparing specificity switching mechanisms in three distinct Cas12a orthologs.
Main Results:
- Cas12a specificity is concentration-dependent, switching with Mg2+ levels.
- Low Mg2+ enhances binding to seed-mutant targets but increases defects from PAM-distal mismatches.
- Different Cas12a orthologs exhibit unique Mg2+-dependent specificity switching, affecting phage escape.
Conclusions:
- Physiological Mg2+ concentrations significantly influence Cas12a specificity.
- The findings highlight the need to consider cellular metal ion conditions for CRISPR-Cas12a tool design.
- Mg2+-dependent specificity switching impacts the evolution of CRISPR-mediated immunity and genome editing efficacy.
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