65 High-fidelity enhanced AsCas12a knock-in mice for efficient multiplexed gene editing, disease modeling and orthogonal immunogenetics

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Background Developing successful immunotherapies heavily relies on the identification of potent and specific targets, and has been a longstanding challenge in the field. It is thus essential to have versatile toolkits enabling efficient endogenous gene editing. The advancement of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology,1 especially the development of Cas9 knock-in mice, has significantly boosted the functional discovery of various genetics factors in diverse fields including genetics, immunology, and oncology.2-5 However, the pleiotropic effects on human disease and the complex nature of gene interaction networks emphasize the need to perform multiplexed gene editing for the discovery of novel therapeutic targets. Methods To facilitate efficient in vivo and ex vivo multiplexed genome engineering, we generated Cre-dependent, conditional LSL-enAsCas12a-HF1 mice by knocking in the enAsCas12a-HF into the Rosa26 locus. We expressed enAsCas12a-HF1 with a CAG promoter, interrupted by a LoxP-3x PolyA Stop-LoxP (LSL) cassette, which allowed conditional enAsCas12a-HF1 expression by Cre recombinase. We then generated constitutive enAsCas12a-HF1 mice by crossing the LSL-enAsCas12a-HF1 mice mice with the CMV-Cre mice.6 To conduct multiplexed gene editing with these mice, we additionally utilized multiple delivery vehicles, including lipid nanoparticles (LNP), adeno-associated virus (AAV), and retroviral vectors. Results By delivering CRISPR RNAs (crRNA) using LNP into the constitutive enAsCas12a-HF1 mice, we achieved functional knockdown of the transthyretin (TTR) protein, a misfolded form of which leads to life-threatening transthyretin amyloidosis.7 Additionally, we demonstrated efficient quadruplex gene knockout in vivo with a single AAV vector simultaneously targeting murine Trp53, Apc, Pten, and Rb1. Injection of this AAV into the the LSL-enAsCas12a-HF1 mice led to rapid induction of salivary gland squamous cell carcinoma and lung adenocarcinoma. Furthermore, we achieved ex vivo multiplexed gene knockout in both primary T cells and bone marrow derived dendritic cells (BMDCs). Notably, we demonstrated the modularity of LSL-enAsCas12a-HF1 mice by integrating with a transgenic mouse line (dCas9-SPH) for gene activation.8 Using BMDCs isolated from the LSL-enAsCas12a; dCas9-SPH mice, we achieved simultaneous gene activation and knockout (DAKO) at the single cell level. Conclusions In summary, we present here new toolkits that compose of the high-fidelity enAsCas12a knock-in mice and associated delivery methods. This system enables versatile capabilities for both multiplexed gene knockout, and simultaneous gene activation and knockout, applicable in both ex vivo and in vivo settings. The flexibility of our system empowers researchers to unravel the complexities of gene function and interaction across various fields of study. References Wang JY, Doudna JA. CRISPR technology: a decade of genome editing is only the beginning. Science 2023;379:eadd8643. Platt RJ, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 2014;159:440-455. Wang G, et al. Mapping a functional cancer genome atlas of tumor suppressors in mouse liver using AAV-CRISPR-mediated direct in vivo screening. Sci Adv 2018;4:eaao5508. Chow RD, et al. AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma. Nat Neurosci 2017;20:1329-1341. Dong MB, et al. Systematic immunotherapy target discovery using genome-scale in vivo CRISPR screens in CD8 T cells. Cell 2019;178:1189-1204 e1123. Schwenk F, Baron U, Rajewsky K. A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic Acids Res 1995;23:5080-5081. Adams D, Koike H, Slama M, Coelho T. Hereditary transthyretin amyloidosis: a model of medical progress for a fatal disease. Nat Rev Neurol 2019;15:387-404. Zhou H, et al. In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR-dCas9-activator transgenic mice. Nat Neurosci 2018;21:440-446.

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