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Updated: May 21, 2025
Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice
Published on: June 24, 2022
Cas12a-knock-in mice for multiplexed genome editing, disease modelling and immune-cell engineering
Kaiyuan Tang1,2,3,4, Liqun Zhou1,2,3,5, Xiaolong Tian1,2,6
1Department of Genetics, Yale University School of Medicine, New Haven, CT, USA.
Abstract:
The pleiotropic effects of human disease and the complex nature of gene-interaction networks require knock-in mice allowing for multiplexed gene perturbations. Here we describe a series of knock-in mice with a C57BL/6 background and with the conditional or constitutive expression of LbCas12a or of high-fidelity enhanced AsCas12a, which were inserted at the Rosa26 locus. The constitutive expression of Cas12a in the mice did not lead to discernible pathology and enabled efficient multiplexed genome engineering. We used the mice for the retrovirus-based immune-cell engineering of CD4+ and CD8+ T cells, B cells and bone-marrow-derived dendritic cells, for autochthonous cancer modelling through the delivery of multiple CRISPR RNAs as a single array using adeno-associated viruses, and for the targeted genome editing of liver tissue using lipid nanoparticles. We also describe a system for simultaneous dual-gene activation and knockout (DAKO). The Cas12a-knock-in mice and the viral and non-viral delivery vehicles provide a versatile toolkit for ex vivo and in vivo applications in genome editing, disease modelling and immune-cell engineering, and for the deconvolution of complex gene interactions.
Insights
Researchers developed novel knock-in mice for advanced genome engineering. These mice enable multiplexed gene editing, aiding in disease modeling and immune cell engineering for complex genetic research.
Area of Science:
- Genetics and Genomics
- Molecular Biology
- Immunology
Background:
- Human diseases often involve complex gene interactions, necessitating advanced tools for genetic manipulation.
- Multiplexed gene perturbations are crucial for understanding intricate gene networks and disease mechanisms.
Purpose of the Study:
- To create and characterize knock-in mouse models for efficient, multiplexed genome engineering.
- To establish a versatile toolkit for in vivo and ex vivo applications in gene editing and disease modeling.
Main Methods:
- Generation of knock-in mice with conditional or constitutive expression of LbCas12a or enhanced AsCas12a at the Rosa26 locus.
- Utilizing these mice for retrovirus-based immune-cell engineering (T cells, B cells, dendritic cells).
- Employing adeno-associated viruses for autochthonous cancer modeling and lipid nanoparticles for liver tissue editing.
Main Results:
- Constitutive Cas12a expression in mice showed no discernible pathology, enabling efficient multiplexed genome engineering.
- Demonstrated successful immune-cell engineering, cancer modeling, and targeted liver tissue editing.
- Developed a novel system for simultaneous dual-gene activation and knockout (DAKO).
Conclusions:
- The developed Cas12a-knock-in mice provide a powerful and versatile platform for diverse genome editing applications.
- This toolkit facilitates ex vivo and in vivo research, including immune-cell engineering and complex gene interaction studies.
- The models are instrumental for advancing disease modeling and understanding the genetic basis of human diseases.
