CasGen: A Regularized Generative Model for CRISPR Cas Protein Design with Classification and Margin-Based Optimization

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated proteins (Cas) systems have revolutionized genome editing by providing high precision and versatility. However, most genome editing applications rely on a limited number of well-characterized Cas9 and Cas12 variants, constraining the potential for broader genome engineering applications. In this study, we extensively explored Cas9 and Cas12 proteins and developed CasGen, a novel transformer-based deep generative model with margin-based latent space regularization to enhance the quality of newly generative Cas9 and Cas12 proteins. Specifically, CasGen employs a strategies that combine classification to filter out non-Cas sequences, Bayesian optimization of the latent space to guide functionally relevant designs, and thorough structural validation using AlphaFold-based analyses to ensure robust protein generation. We collected a comprehensive dataset with 3,021 Cas9, 597 Cas12, and 597 Non-Cas protein sequences from reputable biological databases such as InterPro and PDB. To validate the generated proteins, we performed sequence alignment using the BLAST tool to ensure novelty and filter out highly similar sequences to existing Cas proteins. Structural prediction using AlphaFold2 and AlphaFold3 confirmed that the generated proteins exhibit high structural similarity to known Cas9 and Cas12 variants, with TM-scores between 0.70 and 0.85 and root-mean-square deviation (RMSD) values below 2.00 [A]. Sequence identity analysis further demonstrated that the generated Cas9 orthologs exhibited 28% to 55% identity with known variants, while Cas12a variants show up to 48% identity. Our results demonstrate that the proposed Cas generative model has significant potential to expand the genome editing toolkit by designing diverse Cas proteins that retain functional integrity. The developed deep generative approach offers a promising avenue for synthetic biology and therapeutic applications, enableling the development of more precise and versatile Cas-based genome editing tools.