The humanized transgene was edited and inserted into the mdx mESCs, which are deficient/KO for the mouse DMD gene.

MDX/d2

GRCh38 chrX: 32,713,736_32,875,972 delinsGTTAG

Yes, genotyping is established.

1. Assay designed to confirm the deletion of the entire genomic region. We then sequenced the amplified product by Sanger and reconstructed the precise nucleotide sequence of the newly minted mutant locus.

2. Within the clones confirmed to have the deletion above, perform a 'local' assay around each sgRNA site to check if the second hDMD copy was also edited correctly. This reduced the number of positive clones on average by 90%

3. Within the clones selected in #2, above, perform two assays to confirm that the tail-to-tail portion of the remaining hDMD sequence remained intact. These clones were then expanded, tested for mycoplasma and sent to CRL Embryology for injection.