Extended Data Fig. 7: Confirmation of cell proliferation-promoting function for coPARSE-lncRNAs identified by CRISPR-Cas12a screening and the selection of coPARSE-lncRNA candidates for knockout-rescue assay.
a, Effects of crRNAs targeting the positive control gene RNY1 (left), RP1-212P9.3 (middle) and AL355075.4 (right) on cell proliferation in HeLa cells. Relative confluence of cell proliferation was calculated by normalizing GFP positive percentages at the indicated time points relative to control (day 0). Newly designed paired crRNAs not in the original library are marked with ‘new’. b, Culture images of cell proliferation validation assays for HeLa cells treated with two independent shRNAs for each candidate coPARSE-lncRNA. Scale bars, 200 μm. The experiments were repeated three times with similar results. c, Relative RNA expression of RP1-212P9.3, RP11-563J2.3, and AL355075.4 in different shRNA knockdown assays. d, Design of the crRNA pairs and the PCR primers. Representative images of genomic DNA PCR amplification are shown. ACTIN was used as reference. MW: DNA marker. n = 3 independent biological experiments. e, Knockout efficiency based on PCR results for the targeted genome regions of OPRD1. The genomic DNA amplified by PCR was first normalized to the ACTB locus and then to HeLa cells treated with control AAVS1 crRNAs. f, Representative cell proliferation images for GFP-positive cells. Note that the selected images are all from a fixed field of view. Scale bars, 100 μm. The experiments were repeated three times with similar results. g, High-content imaging assay. The relative cell confluence at the indicated time points was calculated by normalizing to the cell numbers of day 0. h, Comparison of lentivirus packaging efficiency for the original knockout and the knockout-rescue plasmids. The lentivirus packaging efficiency was measured as the infection rates at 3 days post lentivirus infection. Scale bars, 200 μm, Error bars, means ± SD, n = 6 biologically independent experiments, two-sided Student’s t-test, n.s., not significant. i, Successful induction of ectopic gene expression under the indicated induction conditions. j, Scatter plot of the candidate coPARSE-lncRNAs (highlighted in red) selected for the knockout-rescue assay. k, l, Relative RNA expression of targeting genes for the knockout-rescue assays. Two pairs of primers were used to detect (k) endogenous and (l) ectopic expression of RP1-212P9.3 and its homolog or luciferase fragment. For panels a, e, and g, error bars, means ± SD, n = 3 independent biological experiments. For panels c, i, k, and l, n = 2 independent biological experiments.
