Figure 1.

Tiling CRISPR identifies a 5′ region corresponding to a sgRNA cluster, as a potential Xist silencing domain. (a) Upper Panel: Schematic showing reporter system in which Xist induction is inversely correlated with expression of a puromycin resistance gene. Lower Panel: Representative images of Cl36 cells 4 days after culturing in puromycin-containing medium with or without 1 μg/ml doxycycline. Results indicate robust cell death following Xist induction. (b) Screening work flow. (c) Detection of enriched sgRNAs and enriched sgRNA clusters. The top 6 panels show enrichment profiles of individual sgRNAs among indicated samples. Dashed horizontal lines represent a FC level of 1.5. In panels 1–4, 197 sgRNAs that are significantly enriched with maxFC ≥ 1.5 and RSA p < 0.5 in D18^dox+ samples are colored in red and the rest in blue. Among them, 3 sgRNAs are also enriched in D18^dox− samples (highlighted in red in panels 5–6). The 7^th panel shows neighborhood Log₁₀P for each sgRNA within a sliding window. These values were used to identify an sgRNA-enriched cluster (see Methods). The bottom schematic shows a ~2.4 kb Xist region corresponding to an enriched sgRNA cluster, as determined by neighborhood Log₁₀P. Red triangles represent 14 individual sgRNAs used for validation and downstream analysis. (d) Upon being transduced with an indicated sgRNA, cas9-cl36 cells were split into two groups with one group treated with doxycycline (dox+) and the other with DMSO (dox−). After 7 days continued culturing in puromycin, the ratio of the percentage of RFP+ cells in dox+ vs dox− samples were calculated.