TheXISTRNAis a non-codingRNAthat inducesXchromosomeinactivation (XCI). Unlike the mouseXistRNA, how thehumanXISTRNAcontrols XCI in female cells is less well characterized, and its functional motifs remain unclear. To systematically decipher the XCI-involvingelementsofXISTRNA, 11 smallerXISTsegments, including repeats A, D and E;human-specific repeatelements; the promoter; and non-repetitive exons, as well as the entireXISTgene, were homozygously deleted in K562 cells using the Cas9 nuclease and paired guide RNAs at high efficiencies, followed by high-throughputRNAsequencing andRNAfluorescence in situ hybridization experiments. Clones containingenblocand promoterdeletionsthat consistently displayed noXISTRNAs and a global up-regulation ofX-linked genes confirmed that the deletion ofXISTreactivates the inactiveXchromosome. Systematic analyses ofsegmentaldeletionsdelineated that exon 5 harboring the non-repeat element is important forX-inactivation maintenance, whereas exons 2, 3 and 4 as well as the other repeats in exon 1 are less important, a different situation from that of mouseXist. This Cas9-assisted dissection ofXISTallowed us to understand the unique functional domains within thehumanXISTRNA.