Multi-modal single-cell assays provide high-resolution snapshots of complex cell populations but are mostly limited to transcriptome plus an additional modality. Here, we describe Expanded CRISPR-compatible Cellular Indexing of Transcriptomes and Epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell. We demonstrate application of ECCITE-seq to multimodal CRISPR screens with robust direct sgRNA capture and to clonotype-aware multimodal phenotyping of cancer samples. Overall design: All experiments were performed using the 10x genomics V(D)J solution which incorporates scRNA-seq with additional profiling of protein surface markers and sgRNAs (when present). Antibody oligo counts are listed in the ADT and HTO tables and sgRNAs counts in the GDO tables. >>>The raw data for the samples GSM3596095, GSM3596098, GSM3596099, GSM3596100, GSM3596103, and GSM3596104 are being submitted to dbGaP due to patient privacy concerns<<<