An Optimized Cas12a Toolkit for Scalable Combinatorial Genetic Screening and Single-Cell Transcriptomics

CRISPR-based functional genomics enables precise interrogation of gene function and regulatory elements at scale. Recent innovations in CRISPR-Cas technologies have empowered systematic combinatorial perturbation for targeting multiple genes, mapping genetic interactions, and deleting genetic elements, all requiring multiple editing events. Here we present an optimized Cas9-Cas12a CHyMErA platform for combinatorial genetic screening using LbCas12a nuclease and direct repeat (DR) guide scaffold variants. Through systematic optimization screens, we identify D156R LbCas12a as the most efficient nuclease and introduce CHyMErA-Net, deep-learning-based algorithm for predicting Cas12a guide activity. CHyMErA-Net, together with newly derived guide specificity scores, is integrated into a user-friendly online guide selection tool to enhance experimental guide designs. We also describe multiple functional LbCas12a direct repeat variants that can be applied for Cas12a guide multiplexing. Leveraging these innovations, we demonstrate high-efficiency capture of Cas9 and LbCas12a gRNAs in single-cell transcriptomic workflows using customized 10x Genomics protocols. Finally, we extend the system to transcriptional modulation, developing CHyMErA-i (repression) and CHyMErA-a (activation) for combinatorial perturbations in contexts sensitive to DNA damage. Collectively, these advances integrate optimized Cas12a variants, flexible expression platforms, and machine learning-based predictive tools to expand the precision and versatility of combinatorial CRISPR screening.