Direct-seq: employing programmed gRNA scaffold for streamlined scRNA-seq in CRISPR screen

Abstract This protocol aimed to combine the single cell RNA-seq (scRNA-seq) with the CRISPR screening assay based on 10x 3’ RNA-seq platform to profile the transcriptome together with genotype at single cell resolution. To achieve this goal, an A/G mixed capture sequence and tRNA sequence are incorporated into the Tetraloop, Loop2 and Tail of the gRNA scaffold. Therefore, cDNA derived from the gRNA transcripts can be captured and barcoded by poly(dT) primer together with the endogenous mRNA and enriched by the nest PCR. With this method, the CRISPR perturbation and its transcriptional readouts can be profiled together in a streamlined workflow. This method, which we named as Direct-seq, enabled the direct genotyping and expression profiling of the CRISPR screening products at single cell resolution on versatile scRNA-seq platforms across different throughput, including 10x 3’ and 5’ RNA-seq, Fluidigim C1, SMART-seq, BGI DNBelab C4 and others.