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. 2021 Nov 3;16(11):e0252848. doi: 10.1371/journal.pone.0252848

Fig 4. CRISPRi screen reveals several lncRNA loci required for endoderm differentiation.

Fig 4

(A) Correlation of the validation sgRNA score and the screen sgRNA score for all 22 sgRNAs validated, colored by whether the sgRNAs target control mRNA hits (green), lncRNA hits (black), or lncRNA non-hits (white). Validation scores were calculated according to the formula outlined. Example validation data for 8 sgRNAs targeting 2 control mRNA and 2 lncRNA hits are shown in (B)-(F); the full dataset is in S4 Table. r = Spearman’s rho, p = Spearman p-value, n = # sgRNAs. (B) RT-qPCR expression of mRNA SOX17 (sgRNAs #1 and #2) or EOMES (sgRNAs #3 and #4) during endoderm differentiation using either a scrambled negative control sgRNA (light gray bars) or a targeting sgRNA (green bars). Labeled sgRNA numbers correspond to those outlined in (A). *** = p-value < 0.001 by an unpaired t-test. (C) RT-qPCR expression of lncRNA RP11-120D5.1 (sgRNAs #5 and #6) or RP11-222K16.2 (sgRNAs #7 and #8) during endoderm differentiation using either a scrambled negative control sgRNA (light gray bars) or a targeting sgRNA (dark gray bars). Labeled sgRNA numbers correspond to those outlined in (A). * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001 by an unpaired t-test. (D) FACS staining of scrambled negative control sgRNAs during endoderm differentiation, with percentages of differentiated cells (top right box) and undifferentiated cells (bottom left box) labeled. Three separate endoderm differentiation time courses were performed to validate the 8 outlined sgRNAs. Scrambled sgRNA #1 was run as a negative control for targeting sgRNAs #1, #7, and #8; scrambled sgRNA #2 was run as a negative control for targeting sgRNAs #2, #3, and #4; scrambled sgRNA #3 was run as a negative control for targeting sgRNAs #5 and #6. (E) FACS staining of mRNA-targeting sgRNAs #1–4 during endoderm differentiation, with percentages of differentiated cells (top right box) and undifferentiated cells (bottom left box) labeled. (F) FACS staining of lncRNA-targeting sgRNAs #5–8 during endoderm differentiation, with percentages of differentiated cells (top right box) and undifferentiated cells (bottom left box) labeled.