Immunosuppression within the tumor microenvironment (TME) remains a formidable hurdle to effective cancer immunotherapy, largely driven by regulatory T cells (Tregs). Although immune checkpoint blockade has revolutionized cancer treatment, Treg-mediated suppression of effector immune cells can significantly curb its therapeutic impact. To overcome this challenge, we propose a lipid nanoparticle (LNP)-delivered CRISPR gene-editing strategy that selectively reprograms intratumoral Tregs while sparing peripheral Tregs, thus minimizing systemic toxicity. By embedding unique formulation-specific mRNA barcodes within LNPs, we enable high-throughput screening of multiple formulations through a pooled injection in the well-established 4T1 triple-negative breast cancer mouse model. This approach facilitates precise assessments of biodistribution, cellular uptake, gene-editing efficiency, and transcriptional responses in Tregs, as well as therapeutic outcomes. Concurrently, we will develop LCM-LNP-seq, a novel method that couples laser capture microdissection (LCM) with spatial transcriptomics to elucidate how tumor architecture, hypoxia, and vascular proximity influence LNP uptake and function. Insights derived from this integrated platform will guide the rational design of next-generation Treg-targeting therapies, ultimately enhancing anti-tumor immunity in immunologically “cold” tumors while mitigating systemic toxicity.