Multimodal phenotyping in scCRISPR screening and analysis. (A) scCRISPR screening technologies differ in their strategies for capturing gRNAs. Indirect gRNA capture methods use a separate barcode sequence with a poly(A) tail to infer gRNA identity. CROP-seq improves upon this method by integrating the U6 promoter and gRNA cassette into the 3′ long terminal repeat of the lentiviral vector, which is duplicated upon integration, enabling both functional gRNA expression and direct detection of a polyadenylated transcript. In contrast, direct gRNA capture methods allow simultaneous sequencing of gRNAs and transcriptomes using (1) specific primers codelivered with capture beads (5′ strategies), (2) capture sequences linked to oligo-dT beads (3′ strategies), or (3) probe hybridization, as used in the Flex assay. scCRISPR platforms can incorporate diverse readouts, including mRNA expression, chromatin accessibility, V(D)J sequencing of TCRs, and protein abundance. Platforms with multiomic readouts are highlighted. (B) scCRISPR screening of transcription factors paired with transcriptome profiling identifies cofunctional gene modules that regulate differentiation trajectories of CD8⁺ T cell subsets in cancer, including precursor exhausted-like state 1 (Tpex1), Tpex2, terminal exhausted-like state 1 (Tex1), and Tex2 populations. GBC, guide barcode; RT, reverse transcription.