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Characterization of NiCas12b for In Vivo Genome Editing
Yunqian Zhang1, Jingjing Wei2, Hongyan Wang3,4
1Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, State Key Laboratory of Genetic Engineering at School of Life Sciences, Fudan University, Shanghai, 200438, China.
Advanced Science (Weinheim, Baden-Wurttemberg, Germany)
|July 30, 2024
Summary
A novel CRISPR-Cas12b system, NiCas12b, shows efficient genome editing in mammalian cells and successfully lowers cholesterol in mice. This discovery offers a promising new tool for gene therapy and research.
Area of Science:
- Molecular Biology
- Gene Editing Technologies
- Biotechnology
Background:
- The CRISPR-Cas12b system is a powerful tool for genome editing, but its application in human cells and in vivo is limited.
- Identifying novel Cas12b nucleases with high activity and specificity is crucial for advancing gene editing therapeutics.
Purpose of the Study:
- To screen Cas12b orthologs for genome editing activity in mammalian cells.
- To evaluate the therapeutic potential of an identified Cas12b nuclease for in vivo gene editing.
Main Methods:
- A green fluorescent protein (GFP)-activation assay was used to screen 15 Cas12b orthologs.
- NiCas12b was delivered via adeno-associated virus (AAV) to target the Pcsk9 gene in mouse liver.
- Insertion/deletion (indel) efficiency and serum cholesterol levels were measured.
Main Results:
- Four of the 15 screened Cas12b orthologs showed editing activity in mammalian cells.
- NiCas12b, from Nitrospira sp., demonstrated efficient genome editing with a "TTN" protospacer adjacent motif (PAM) and high specificity.
- In vivo delivery of NiCas12b resulted in over 16.0% indel efficiency in the mouse liver, significantly reducing serum cholesterol levels.
Conclusions:
- NiCas12b is a highly efficient and specific CRISPR-Cas12b nuclease suitable for therapeutic applications.
- This study validates NiCas12b as a novel tool for both basic research and clinical gene editing, particularly for conditions like hypercholesterolemia.
