Hyeon J. Lee1,†, Ramu Gopalappa2,†, Hongjae Sunwoo3,†, Seo-Won Choi1, Suresh Ramakrishna4,5, Jeannie T. Lee3, Hyongbum H. Kim2,6,7,8,* and Jin-Wu Nam1,9,*
1 Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 04763, Republic of Korea, 2 Department of Pharmacology, Yonsei University College of Medicine, Seoul 03722, Republic of Korea, 3 Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, Boston MA 02114, USA, 4 Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, Republic of Korea, 5 College of Medicine, Hanyang University, Seoul 04763, Republic of Korea, 6 Brain Korea 21 Plus Project for Medical Sciences, Yonsei University College of Medicine, Seoul 03722, Republic of Korea, 7 Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03722, Republic of Korea, 8 Center for Nanomedicine, Institute for Basic Science (IBS), Seoul 34126, Republic of Korea and 9 Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul 04763, Republic of Korea
*To whom correspondence should be addressed.
Correspondence may also be addressed to Hyongbum Henry Kim.
†The authors wish it to be known that, in their opinion, the first three authors should be regarded as Joint First Authors.
The XIST RNA is a non-coding RNA that induces X chromosome inactivation (XCI). Unlike the mouse Xist RNA, how the human XIST RNA controls XCI in female cells is less well characterized, and its functional motifs remain unclear. To systematically decipher the XCI-involving elements of XIST RNA, 11 smaller XIST segments, including repeats A, D and E; human-specific repeat elements; the promoter; and non-repetitive exons, as well as the entire XIST gene, were homozygously deleted in K562 cells using the Cas9 nuclease and paired guide RNAs at high efficiencies, followed by high-throughput RNA sequencing and RNA fluorescence in situ hybridization experiments. Clones containing en bloc and promoter deletions that consistently displayed no XIST RNAs and a global up-regulation of X-linked genes confirmed that the deletion of XIST reactivates the inactive X chromosome. Systematic analyses of segmental deletions delineated that exon 5 harboring the non-repeat element is important for X-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 mouse Xist. This Cas9-assisted dissection of XIST allowed us to understand the unique functional domains within the human XIST RNA.