Pathway Sculptor for Compact and Versatile Combinatorial Genetic Perturbation

The ability to perturb multiple proteins simultaneously within the same cell is essential for understanding and re-engineering biological pathways. CRISPR-Cas12a mutants with inactivated DNAse but intact RNAse activity (dCas12a) retain the ability to process large CRISPR RNAs (crRNAs) arrays, enabling them to target multiple genomic loci in parallel. When coupled with transcriptional effector domains, these properties make Cas12a a promising platform for multi-locus transcriptional perturbation. However, current Cas12a-based CRISPRi systems exhibit limitations in processing of multi-crRNA arrays and transcriptional regulation. Here, we combine molecular and circuit-level engineering to develop a programmable Cas12a- based CRIPSRi system capable of strong, tunable, and simultaneous knockdown of six or more genes in a single cell without genomic DNA cleavage. We demonstrate the utility of this system by systematically perturbing a partially redundant set of Bone Morphogenetic Protein (BMP) receptors, enabling quantitative analysis of BMP signaling across diverse receptor configurations.