Sequencing of genetic perturbations at single-cell resolution enables the precise profiling of transcriptional effects but lacks the ability to examine their impact on tissue environments. Here, we present Perturb-DBiT, a platform that allows for the simultaneous co-profiling of spatial transcriptomics and single-guide RNAs (sgRNAs) in tissue with genome-scale CRISPR libraries, offering a comprehensive understanding of how genetic modifications affect cellular behavior and tissue architecture in vivo. This versatile platform supports a variety of viral delivery vectors, sgRNA library sizes, and tissue preparations including formalin-fixed paraffin-embedded (FFPE) samples, along with two distinct sgRNA capture methods, making it adaptable to a vast range of experimental designs. By leveraging Perturb-DBiT, we conducted unbiased knockouts of tens of genes or at genome-wide scale across different cancer models, revealing insights into clonal cooperation and dynamics. The platform’s capability to map sgRNA species in individual colonies while concurrently mapping the transcriptome from the same sample allows for a detailed and integrative analysis of genetic interactions and molecular mechanisms within and between single cells, within their spatially distinct regions. Perturb-DBiT is particularly effective in assessing the effects of genetic perturbation on tumor immune microenvironments (TIME), thereby uncovering differential and synergistic interactions that may promote tumor infiltration or suppression. This approach ultimately enables the identification of cancer-specific essential genes as potential therapeutic targets and provides a rich framework for studying perturbations that drive tumor initiation, progression, and therapeutic response in single cells. Characterizing these mutations by their dynamic spatial organization alongside other omics methods such as spatial proteomics offers a deeper understanding of the complex structural dynamics of disease. Ultimately, Perturb-DBiT broadens the scope of genetic inquiry and lays the groundwork for developing precision therapy, illuminating where and how mutations occur in tissues and their impact on cellular processes.