Direct detection of CRISPR mutations and transcriptional responses at single cell resolution in vivo

Abstract

CRISPR screens coupled with single-cell RNA sequencing are transforming high-throughput functional genomics. However, applications in vivo remain limited and are confounded by difficulties in identifying and genetically characterizing edited cells. Here we present scPT-seq, a single-cell RNA assay that resolves CRISPR-induced mutations at base-pair resolution and captures transcriptional responses in the same single cells in vivo. scPT-seq comes with a computational analysis suite enabling haplotype-resolved mutation detection and characterization of complex editing outcomes, including splice-junction variation. Applied to the Drosophila intestine, a highly regenerative tissue, scPT-seq distinguishes cell-autonomous from environmental effects by identifying mutant and wild-type cells within tissues, and reveals spatially organized compensatory mechanisms in response to mutations. By using editing outcomes as heritable clonal markers, we identified distinct intestinal stem cell populations with specialized differentiation trajectories. In summary, scPT-seq provides a versatile technology for dissecting gene function and lineage dynamics in complex tissues.