Journal: bioRxiv
Article Title: Direct capture of CRISPR guides enables scalable, multiplexed, and multi-omic Perturb-seq
doi: 10.1101/503367
Figure Lengend Snippet: (a) Schematic of 3’ single-cell RNA-sequencing (3’ scRNA-seq). Polyadenylated mRNAs from individual cells (top, light blue) anneal to barcoded oligo-dT primers in emulsion droplets (delivered to droplets on gel beads) and are reverse transcribed into indexed cDNA (bottom). TSO, template switch oligo. UMI, unique molecular identifier. CBC, cell barcode. (b) Schematic of experimental workflows for GBC or direct capture Perturb-seq (3’ or 5’) based on protocols from 10x Genomics. Red indicates generation of sequencing libraries. Box details construction of index sequencing library for GBC Perturb-seq, which is based on our previously published protocol . (c) CRISPRi activity of guides carrying the indicated capture sequences (all programmed with the GFP-targeting protospacer EGFP-NT2) in GFP+ K562 dCas9-KRAB cells 6 or 10 days post-transduction. Data from guides selected for direct capture experiments (sgRNA cs1 and sgRNA cs2 ) are indicated. 30 (A) indicates 30 adenines. For comparison, data from standard guides also programmed with EGFP-NT2, without capture sequences, and expressed from 3 other vectors (indicated) were also included. One of these, indicated as “CROP-seq,” has a different (previously published) constant region and is expressed from a different promoter. Data was collected in three independently controlled experiments and represents the average of triplicate measurements normalized to control measurements ± standard error. For reference, the median GFP of transfected controls (relative to the median of untransfected cells in the same well) were 0.14, 0.14, and 0.11. (d) Gaussian kernel density estimates of normalized flow-cytometry measurements representing GFP expression demonstrate CRISPRi activity of the indicated guide RNAs (programmed with the GFP-targeting protospacer EGFP-NT2). The negative and positive control data are the same as in . (e) Schematic of 5’ single-cell RNA-sequencing (5’ scRNA-seq). Polyadenylated mRNAs from individual cells (top, light blue) anneal to unbarcoded oligo-dT primers in emulsion droplets (delivered to droplets as free oligos) and are reverse transcribed. Indexing of cDNA (bottom) occurs after template switching allows for extension of barcoded TSOs (delivered to droplets on gel beads).
Article Snippet: Two vectors evaluated in (pBA950 labeled as “CROP-seq modified for CRISPRi” and pBA960 labeled as “CROP-seq”) were derived from CROPseq-Guide-Puro (Addgene, #86708).
Techniques: RNA Sequencing Assay, Sequencing, Activity Assay, Transduction, Transfection, Flow Cytometry, Expressing, Positive Control
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