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. Author manuscript; available in PMC: 2020 Apr 4.
Published in final edited form as: Mol Cell. 2019 Feb 28;74(1):101–117.e10. doi: 10.1016/j.molcel.2019.01.015

Figure 1. CRISPR/Cas9 deletion screen identifies Xist functional domains.

Figure 1.

See also Figures S1-S3.

(A) Diagram of Xist locus, repeat elements, gRNA target sites, and qPCR amplicons.

(B) Schematic of screening method using tandem two-color RNA FISH.

(C) Xist RNA FISH for deletions showing altered Xist cloud morphology in Xi+/− MEFs. Arrowhead indicates WT and arrow indicates mutant Xist cloud. Right panels show 3x zoom-in of each cloud.

(D) RT-qPCR showing effect of deletions on Xist RNA levels in Xi−/− MEFs. Error bars show standard deviation for 3 biological replicates.

(E) 3D STORM imaging and size measurements of Xist clouds in ΔRepB and ΔRepE Xi+/− MEFs. Epifluorescent images of same cells shown to the right, with arrowhead indicating WT and arrow indicating mutant Xist cloud. p-values by two-tailed t-test.

(F) H3K27me3 and H2AK119ub IF for deletions showing phenotype in Xi+/− MEFs. Arrowhead indicates WT and arrow indicates mutant Xist cloud.