Extended Data Figure 8: RNAP II pausing defect and R-loop accumulation are observed in CRISPR SMN knockout cells and in SMA disease cells.

a, ChIP for POLR2A with N20 antibody was performed after stable SMN knockout (CRISPR) in HEK293 cells shows that RNAP II accumulates in the termination regions of ACTB. Scrambled guide RNA treatment was used as a negative control. Error bars represent biological variation (mean ± s.e.m., n = 3). b, Accumulation of R-loops in the termination regions of the ACTB gene after SMN knockout. A fusion protein of GFP–RNase H DNA–RNA hybrid binding ___domain was stably expressed in HEK293 cells. ChIP with GFP antibody (Abcam 290) was used for the detection of the R-loops (DNA–RNA hybrids), using the indicated primer positions for qPCR along the gene. Scrambled guide RNA treatment was used as a negative control. Error bars represent biological variation (mean ± s.e.m., n = 3). c, Top: live cell microscopy images showing that HEK293 cells with SMN knockout appear to be physiologically normal in comparison to the control scrambled KO. Bottom: western blot with anti-SMN antibody showing that SMN expression is knocked out by CRISPR. d, Human cell lines (3 fibroblast, 3 B lymphocyte) were obtained from the Coriell Institute for Medical Research. These include cells from two children with SMA disease and their normal parents. e, ChIP for POLR2A (N20, 8WG16 antibodies) was performed on the ACTB gene using the averaged value of the parents as control. POLR2A in the SMA disease cells (from both fibroblast and B cell lines) accumulates in the termination regions of the ACTB gene. Error bars represent biological variation (mean ± s.e.m., n = 4). f, Top: quantification of R-loops by DNA immunoprecipitation (DIP) with the S9.6 antibody in the patient cells, showing that the R-loops are sensitive to RNase H. Error bars represent technical variation (mean ± s.e.m., n = 3). Bottom: R-loop DIP with the S9.6 antibody shows that R-loops accumulate in the termination regions of the ACTB gene in the SMA disease cells. The averaged value of the parents was used as a control. Error bars represent biological variation (mean ± s.e.m., n = 5).