Fig. 1: Design of a genome-editing strategy to disrupt the Obl mutant allele.
a SpCas9 sgRNAs were designed to target the mutant Atp2b2 Obl allele, in which C2765 is changed to T (red). The protospacer (green arrows) of each Obl-targeting sgRNA contains a complementary A or T (red) that pairs with the mutation in the Obl allele, but that forms a mismatch with wild-type Atp2b2 allele. b In vitro Cas9:sgRNA-mediated Atp2b2 DNA cleavage. 50 nM of a 956-bp DNA fragments of Atp2b2 or the Atp2b2Obl/+ was incubated with 300 nM of each of the three Cas9:sgRNA for 15 min at 37 °C. Expected cleavage products of 553-bp and 403-bp were detected in all samples. M = 100 bp ladder. This was repeated independently for 3 times with similar results. c Quantification of DNA cleavage in (b) by densitometry using imageJ. d Editing shown by the indel percentages in three unsorted adult mouse primary fibroblast cells (WT, Atp2b2Obl/+ and Atp2b2Obl/Obl) after nucleofection of the RNP complex of Cas9:Atp2b2-mut1 gRNA. Indels were only detected in the cells with the Obl allele. n = 3 biologically independent experiments. Values and error bars are mean ± SD. Source data are provided as a Source data file. e NGS reads of indel from (d). The Obl mutation was highlighted in yellow. The red arrow indicates the cutting site. The list of indels is not comprehensive, only the top representative indels are shown.
