Fig. 2

The demonstration of the programmed scaffold in single-cell CRISPR screen. a Two tRNA sequences from human (Gln) and rice (Gly) were incorporated into the gRNA expression cassette, locating at the downstream of the U6 promoter. The editing efficiencies were examined for gRNA scaffolds with the capture sequence inserted at the tail, tetraloop, and loop2 (*P ≤ 0.05, **P ≤ 0.01, compared with U6 in 2-way ANOVA test followed by Tukey’s multiple comparisons test). b Workflow of the library preparation using the 10x 3′ single-cell RNA-seq kit. Two index gRNA libraries were generated from the pre-amplified cDNA via nested PCR. Half of the cDNA was directly used as the PCR template; the other half was size selected as described in the 10x protocol. The 1st PCR of the nested PCR used a tRNA-specific primer to enrich gRNA-derived cDNA. The 2nd PCR added the P5 and P7 to enable NGS sequencing. The mRNA library was prepared as stated in the 10x protocol. c Histogram of the gRNA UMI per cell from the index gRNA library. d The tSNE cell clustering of the 10x data