Fig. 3: CRISPR–Cas12a screening and validation of coPARSE-lncRNA functions.

a, The crRNA library was delivered into cells stably expressing Cas12a by lentiviral infection. Infected cells were collected by fluorescence-activated cell sorting (FACS; green fluorescence). For screening, cells were cultured for 15–45 d before genome DNA extraction and high-throughput sequencing analysis of the barcoded crRNA regions. Each DNA oligonucleotide sequence encodes two crRNAs (represented in red and blue), which will be transcribed and processed to generate individual mature crRNAs by Cas12a; these mature crRNAs will guide Cas12a to cut target genome regions. DR (19 nt). b, The RRA scores for the top-ranking negatively selected lncRNAs. Note that smaller RRA scores indicate a stronger selection of the corresponding lncRNAs. The coPARSE-lncRNAs of the top ten negatively selected lncRNAs are highlighted in red, whereas the non-coPARSE-lncRNAs are highlighted in orange. Nine positive control genes are shown in blue (round dots for lncRNAs and triangles for protein-coding genes). Background represents the overall distribution. c, The mean read count value for paired crRNAs at day 45 relative to that of day 0 for lncRNA genes in our screening library. Highlighted dots are paired crRNAs for five negatively selected candidate genes in our screening assay, and the background represents the overall distribution. d, Overlap of the negatively selected lncRNAs in the three indicated cell lines. e, Cell proliferation validation assays in HeLa cells treated with two independent shRNAs for each candidate lncRNA. Error bars, means ± s.d., n = 3 biologically independent experiments. DR, direct repeats.