Alternative splicing of mRNA precursors results in multiple protein variants from a single gene and is critical for diverse cellular processes and development. Xist encodes a long noncoding RNA which is a central player to induce X-chromosome inactivation in female mammals and has two major splicing variants: long and short isoforms of Xist RNA. Although a differentiation-specific and a female-specific expression of Xist isoforms have been reported, the functional role of each Xist RNA isoform is largely unexplored. Using CRISPR/Cas9-mediated targeted modification of the 5′ splice site in Xist intron 7, researchers from Cincinnati Children’s Hospital Medical Center create mutant female ES cell lines which dominantly express the long- or short-splicing isoform of Xist RNA from the inactive X-chromosome (Xi) upon differentiation. Successful execution of CRISPR/Cas-based splicing modulation indicates that their CRISPR/Cas-based targeted modification of splicing sites is a useful approach to study specific isoforms of a transcript generated by alternative splicing. Upon differentiation of splicing-mutant Xist female ES cells, the researchers found that both long and short Xist isoforms can induce X-chromosome inactivation normally during ES cell differentiation, suggesting that the short splicing isoform of Xist RNA is sufficient to induce X-chromosome inactivation.
Short and long Xist isoform-specific targeting with CRISPR/Cas9
(A) Alignment of all 5′ and 3′ splice sites in Xist with a consensus splicing sequence of a major U2 class intron (36,37). r: adenine (a) or guanine (g); y is cytosine (c) or thymine (t). The exon and intron nucleotide sequences are capitalized or lowercased, respectively. Arrowheads indicate cleavage sites by splicing. (B) Map of Xist/Tsix locus to show the strategy of isoform-specific targeting and the Tsix truncation. The asterisk indicates the position of primer pair (X7) used in (C). The sgRNA sequence and adjacent protospacer-adjacent motif (PAM) for CRISPR/Cas9 genome editing are shown as green and orange boxes, respectively. The mutations introduced by CRISPR/Cas9 are labeled in red. Arrow indicates double-strand break site. HindIII site to confirm CRISPR/Cas9-based targeted HDR is highlighted by blue. SA, splicing acceptor; IRES, internal ribosome entry site; Hyg, hygromycin resistance gene; tpA, tandem polyadenylation signal. (C) Genomic PCR and following HindIII digestion analysis to confirm the CRISPR/Cas9 targeting. (D) qRT-PCR of short and long Xist expression in ES cells to confirm the splicing efficiency alteration in isoform-specific targeting cell lines. Gapdh was used as an internal control for normalization. The mean ± SD from three independent experiments is shown. P-values were calculated to TST control by an unpaired t-test (*P < 0.05, **P < 0.01, ***P < 0.001).