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. 2016 Nov 15;1:13. [Version 1] doi: 10.12688/wellcomeopenres.10011.1

Figure 2. Generation of MeCP2 knock-out LUHMES cell lines.

Figure 2.

( A) Schematic representation of the MECP2 locus with the targeting sequence of sgRNA A and sgRNA B labelled. Arrowheads indicate sites of double-strand break. ( B) Schematic representation of the plasmid used to deliver Cas9 and sgRNA to LUHMES cells. ( C) T7E1 assay of sgRNA A and sgRNA B. WT: non-transfected wild-type cells. NDC: wild-type cells Nucleofected without any DNA. Asterisks indicate non-specific bands. Arrows indicate bands specific to samples containing Cas9 and sgRNA. ( D) Sequencing of genomic DNA from two LUHMES single-cell clones. Wild-type genomic DNA is in lower case at the top. ( E) Sequencing of cDNA from two LUHMES single-cell clones. Wild-type cDNA is in lower case at the top. ( F) Sequencing of genomic DNA from a single LUHMES unedited clone that was transfected with Cas9 and sgRNA B. Wild-type gDNA is in lower case at the top. ( G) Western blot of MeCP2 protein and Histone H3 loading control from wild-type cells (WT), wild-type cells that went through the Nucleofection and cloning process (WTC), KO1 and KO2 cell lines. ( H) Sequencing of sgRNA off-target sites in KO1 and KO2 cell lines. Numbers next to each locus name indicate the off-target score as determined by crispr.mit.edu.