Design and analysis of CUL9 KO hPSC clones.

( A ) Sequence of CUL9 exon 9 with antisense gRNA aligned to target sequence. Figure made using Benchling ( B ) CUL9 KO clones have single nucleotide insertions resulting in a frameshift mutation. Table summarizing the results of Next Generation Sequencing (NGS) analysis of control and CUL9 KO lines used for downstream experiments. Segment of CUL9 exon 9 sequenced for analysis by NGS are shown below table for each cell line. gRNA targeted sequence is highlighted in yellow, and single nucleotide insertions are highlighted in green. ( C ) CUL9 KO cells do not express CUL9 protein or increased levels of homologue CUL7. Western blot of analysis of control and CUL9 KO clones to analyze CUL9 and CUL7 protein levels. Isogenic WT and Clone #1 n = 4; Clone #2 n = 3; mean +/- SEM; Analysis done using student’s t-test, α = 0.05. (TIF)