Extended Data Fig. 1.

a. The vector system used in PerturbSci for dCas9 and sgRNA expression. The expression of the enhanced CRISPRi silencer dCas9-KRAB-MeCP2⁹ was controlled by the tetracycline responsive (Tet-on) promoter. A GFP sequence was added to the original CROP-seq-opti plasmid⁶ as an indicator of successful sgRNA transduction and for the lentivirus titer measurement. The CROP-seq vector utilizes the self-replication mechanism of lentivirus during the integration for amplifying the sgRNA expression cassette. In this lentiviral plasmid, the sgRNA expression cassette replaced the U3 region of the 3’LTR⁵. During the lentiviral integration, the shortened 5’LTR of reverse-transcribed lentiviral genomic DNA was extended by using its 3’LTR as a template, and the sgRNA expression cassette is self-replicated to the 5’LTR⁶². The self-replicated sgRNA expression cassette at 5’LTR generates functional sgRNA for guiding dCas9, and the original expression cassette at 3’LTR is transcribed as a part of the Puro-GFP fusion transcript driven by the EF-1α promoter. b. The library preparation scheme and the final library structures of PerturbSci, including a scalable combinatorial indexing strategy with direct sgRNA capture and enrichment that reduced the library preparation cost, enhanced the sensitivity of the sgRNA capture compared to the original CROP-seq⁵, and avoided the extensive barcodes swapping detected in Perturb-seq⁶. c. A schematic comparison of chemistries between PerturbSci, CROP-seq⁵, and Direct-capture Perturb-seq⁷.