CROPseq-multi: a versatile solution for multiplexed perturbation and decoding in pooled CRISPR screens

Forward genetic screens seek to dissect complex biological systems by systematically perturbing genetic elements and observing the resulting phenotypes. While standard screening methodologies introduce individual perturbations, multiplexing perturbations improves the performance of single-target screens and enables combinatorial screens for the study of genetic interactions. Current tools for multiplexing perturbations are incompatible with pooled screening methodologies that require mRNA-embedded barcodes, including some microscopy and single cell sequencing approaches. Here, we report the development of CROPseq-multi, a CROPseq-inspired lentiviral system to multiplex Streptococcus pyogenes (Sp) Cas9-based perturbations with mRNA-embedded barcodes. CROPseq-multi has equivalent per-guide activity to CROPseq and low lentiviral recombination frequencies. CROPseq-multi is compatible with enrichment screening methodologies and optical pooled screens, and is extensible to screens with single-cell sequencing readouts. For optical pooled screens, an optimized and multiplexed in situ detection protocol improves barcode detection efficiency 10-fold, enables detection of recombination events, and increases decoding efficiency 3-fold relative to CROPseq. CROPseq-multi is a widely applicable multiplexing solution for diverse SpCas9-based genetic screening approaches.