hDMD del45 mdx and hDMD del45 mdxD2 mice were electroporated as described [12 (link)] with 20µg of px333 plasmid DNA (Addgene 64073, Andrea Ventura [13 (link)]) containing CRISPR guides 44C4 and 55C3 (from [11 (link)]) or pmaxGFP as a control forhDMDdel45 mdxD2mice. In brief, 5µl hyaluronidase was injected into the flexor digitorum brevis (FDB) muscle and 1hr later the DNA was injected and electroporated 20 times for 20 ms at 1 Hz.
hDMD del45 mdx mice were harvested 22 or 33 days later and genomic DNA was extracted by digesting the muscles with proteinase K then using the Quick-gDNA™ Miniprep Kit (Zymo Research). PCR for an exon 45–55 deletion was performed as described using Accuprime Taq HiFi (Thermo Fisher Scientific) or Herculase II Fusion Polymerase (Agilent Genomics) [11 (link)]. Sequencing of blunt cloned PCR products from Zero Blunt® TOPO® (Life Technologies) was done by Laragen Inc.
hDMD del45 mdxD2 mice were harvested 24 days post-electroporation. The interosseous (IO) and FDB were flash frozen and samples of 10µm cryosections taken throughout the whole muscle. Intervening sections as well as the lumbricalis were used for genomic DNA extraction and PCR as above.
Please see expandedMaterials and Methods in the Supplementary Data .
hDMD del45 mdx mice were harvested 22 or 33 days later and genomic DNA was extracted by digesting the muscles with proteinase K then using the Quick-gDNA™ Miniprep Kit (Zymo Research). PCR for an exon 45–55 deletion was performed as described using Accuprime Taq HiFi (Thermo Fisher Scientific) or Herculase II Fusion Polymerase (Agilent Genomics) [11 (link)]. Sequencing of blunt cloned PCR products from Zero Blunt® TOPO® (Life Technologies) was done by Laragen Inc.
hDMD del45 mdxD2 mice were harvested 24 days post-electroporation. The interosseous (IO) and FDB were flash frozen and samples of 10µm cryosections taken throughout the whole muscle. Intervening sections as well as the lumbricalis were used for genomic DNA extraction and PCR as above.
Please see expanded
