Figure 5.

CRISPR-cas9 mediated mutations and deletion of almost the entire genomic locus of Sema4B do not effect proliferation. (A) Map of the genomic locus of Sema4B showing the potential alternative splicing, location of the gRNA targets and PCR targets used for the analysis of the genomic locus after CRISPR-cas9 mediated mutations or deletion. (B) Representative western blot of U87-GM cell pools treated with sh-cont1 or shRNA1 or 2. In addition protein extracted from cell clones generated by CRISPR-cas9 and cont1 gRNA (clone1), gRNA1 (clone 1) or gRNA 2 (clone 2) targeting the signal sequence of Sema4B are shown. The full-length blots are presented in Supplementary Fig. 4. (C) qPCR analysis of the same CRISPR cell clones demonstrate a variable effect on mRNA expression of individual clones. (D) Resazurin cell viability assay was used to evaluate the CRISPR clones in cell proliferation assay. Cells were tested one day after plating and four days in culture. The fluorescent read on day 4 was normalized to the read of the same cell line on day 1. No effects on proliferation are detected. (E) Representative western blot of U87-GM cell clones generated by two rounds of CRISPR-cas9 with two sets of gRNA in each round (the full-length blots are presented in Supplementary Fig. 4). One control line (cont2) and two deletion lines are shown (del1 and del2). Two cell clones were negative for oligo sets c,d and e,f (both were positive for fragment a,b) qPCR analysis of the same del1 and del2 cell clones show that mRNA levels of both clones are very low with del 2 almost undetectable. (G) Resazurin cell viability assay was used to evaluate del1 and del2 clones in cell proliferation assay. Cells were tested one day after plating and four days in culture. The number of cells on day 4 was normalized to the same cell line on day 1. No effects on proliferation are detected.