Fig. 2: Pooled screen of SmOPT and U7 hairpin variants using split-pool single cell barcoding. | Nature Communications

Fig. 2: Pooled screen of SmOPT and U7 hairpin variants using split-pool single cell barcoding.

From: An engineered U7 small nuclear RNA scaffold greatly increases ADAR-mediated programmable RNA base editing

Fig. 2

a Mutagenesis screen workflow. 1) Three antisense guide RNAs targeting the RAB7A 3’UTR, SNCA 3’UTR, or GAPDH CDS were each cloned onto 252 mutagenesis variants along the Sm binding or U7 hairpin sequences, in duplicate pools. 2) Plasmid libraries were sequenced to match the guide RNA with a 14 N random GuideID barcode located in the GFP 5’UTR. 3) Plasmid libraries were integrated into the Landing Pad 293T cell line. 10 M cells, each containing a single copy of construct, were pooled, fixed, and permeabilized. 4) Four custom primers reverse transcribed GFP, RAB7A, SNCA, or GAPDH transcripts within each fixed cell. Each RT primer possessed an additional 10 N umi and an anchor sequence, which was annealed to a Splint scaffold. 5) Cells were split equally across 60 different wells, each containing a different 7mer subcode that was ligated onto the reverse transcribed cDNA. Cells were then washed, pooled, and split again across 60 new subcode wells. For 60 subcodes, 3 rounds of split-pool ligation yields 216,000 possible combinations. Finally, labeled cells were split into separate wells of 10,000 cells each, lysed, and amplified with dual index primers for Illumina sequencing. b For each of the 750+ guide RNAs, RNA editing was measured for all three transcripts. Editing results comparing the duplicate plasmid libraries (Replicate Pools A versus B) are shown. Guide RNAs targeting RAB7A, SNCA, or GAPDH are shown in red, yellow, or blue, respectively. Positive and negative controls for editing are marked by black symbols. c For each gene target, the effect on RNA editing of single nucleotide substitutions across the SmOPT sequence or paired substitutions across the mU7 hairpin is shown. Underscores indicate locations where extra nucleotides were inserted. In some cases, two nucleotides were inserted. The multicolor dashed line marks the percent editing from the original SmOPT mU7 hairpin construct. Left panels show RNA editing rates from the pooled screen; right panels show RNA editing rates when desired mutations were individually validated at a single copy in the Landing Pad cell line. Source data are provided as a Source Data file.

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