Cas12a-knock-in mice for multiplexed genome editing, disease modelling and immune-cell engineering

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.