Animal studies

All procedures and experiments using mice were approved by the South Australian Health and Medical Research Institute (SAHMRI) Animal Ethics Committee and conducted per the Australian code for the care and use of animals for scientific purposes.

Mice were housed in 13.5-h light/10.5-h dark cycles and were fed the Teklad Global 2918 rodent diet ad libitum. Except where indicated, male mice were used in characterisation experiments. Tg(DMD)72Thoen/J (hDMDTg) were obtained from JAX (stock #018900) and maintained in-house [8]. mDmdKO (ΔEx51) and hDMDTgSc were generated as described in the sections below and maintained in-house. We maintain hDMDTgSc mice as a tick-over colony of homozygotes. C57Bl/6 J mice are maintained in house (JAX stock #000664).

Mouse generation

To create the intervening deletion of a single copy of the hDMD transgene, we designed SpCas9 gRNAs targeting the Neomycin and Hygromycin antibiotic resistance cassettes in the integrated YAC sequence. Complementary gRNA oligos with overhangs facilitating golden gate assembly were phospho-annealed and inserted into pSpCas9(BB)-2A-Puro (PX459) V2.0 [25]. pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62,988; http://n2t.net/addgene:62988; RRID:Addgene_62988). Following plasmid validation by BbsI diagnostic digest and Sanger sequencing (Australian Genome Research Facility), gRNAs were amplified using NEB Phusion in HF buffer with primers containing a 5’ T7 promoter sequence. PCR amplicons purified using the QIAquick PCR Purification Kit (Qiagen) were used to generate gRNA using the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB) and purified using the RNeasy Mini Kit (Qiagen).

hDMDTgTg/Tg mouse sperm was used for in vitro fertilisation of C57BL/6 J oocytes, and 2PN embryos were injected with a microinjection mix containing a final concentration of 100 ng/μL SpCas9 mRNA and 50 ng/μL each of NeoR and HygR gRNA. This was followed by oviduct transfer into pseudo-pregnant females. Intervening transgene deletion was detected in founders by PCR amplification using the forward NeoR and reverse HygR primers.

mDmdKO mice were generated by deletion of Dmd Exon 51 via CRISPR microinjection of C57BL/6 J embryos, as previously described [26].

DNA PCR and Sanger sequencing

Genomic DNA (gDNA) was isolated from mouse ear punch biopsies using the PCR Template Preparation kit (Roche). Targets were PCR amplified using Taq Polymerase (Roche) and FailSafe™ Buffer J (Epicentre) and resolved on a 1% agarose TBE gel.

To prepare samples for sequencing, PCR reactions were cleaned up using the QIAquick PCR Purification kit (Qiagen). Samples with sequencing primers added were sent to the Australian Genome Research Facility (AGRF) for the purified DNA Sanger sequencing service.

Short-read genome sequencing

Library prep and sequencing was performed as a service by the Australian Genome Research Facility (AGRF). Genomic DNA extracted from hDMDTg mice liver were sheared and PCR-free sequencing libraries prepared. From these libraries, 150 base pair, paired-end reads were generated on a NovaSeq 6000. Image analysis was performed in real time by the NovaSeq Control Software (NCS) v1.7.5 and Real Time Analysis (RTA) v3.4.4. Then the Illumina bcl2fastq v2.20.0.422 pipeline was used to generate the final fastq files.

Mapping

All mapping of short read data was carried out using BWA-MEM v 0.7.17 [27] setting option -K, the number of input bases, to 100,000,000 per batch and all other options as default.

A custom reference genome was prepared which combined the GRCm38.p3 mouse genome (GenBank: GCA_000001635.5), a segment of human chromosome X (NCBI RefSeq: NC000023.11; chrX:31,081,740–33,507,715) and pYAC4 (GenBank: U01086.1). The chrX segment was defined by identifying the extents of reads accurately mapped to the region flanking DMD on chrX in the hs38DH human genome (created using the run-gen-ref script from bwakit) [28]. The pYAC4 vector sequence was used as a proxy for the pRANT-11 and pLGTel-1 vectors that were originally used for cloning of the hDMD YAC transgene and for which no sequence data are available [29].

Alignments were sorted by genome coordinate positions using samtools sort v 1.17 and duplicate reads were marked using sambamba markdup v 0.8.2 [30].

Variant calling

Structural variants were called using GRIDSS v 2.12.0 with default settings [31].

Oxford nanopore genome sequencing

High molecular weight genomic DNA was extracted from homozygous hDMDTg and hDMDTgSc mice liver using the Monarch HMW DNA Extraction Kit for Tissue (NEB). Oxford Nanopore Technologies Promethion genome sequencing was performed as a service by Novogene, (Novogene HK). Sequence libraries were prepared using the DNA ligation sequencing protocol with the SQK-LSK-110 kit. Sequence data were generated using a Promethion 9.4.1 flow cell. Bases were called with bonito v0.6.2 (Oxford Nanopore Technologies) with the dna_r9.4.1_e8_sup@v3.3 super accurate base calling model.

Mapping

Oxford Nanopore long-read data were mapped to the custom GRCm38 + DMD + pYAC4 genome described above and also the human genome reference GRCh38 using minimap2 v2.17-r941 with default parameters for nanopore sequencing.

Variant calling

Structural variants were called from mapped long-read genome sequencing with Sniffles2 using default settings [32].

Assembly

Nanopore sequence reads were assembled de novo using Shasta v 0.11.1 [33] using the Nanopore-May2022 model adjusting the minimum length of input reads to 5 kb. To identify contigs associated with the hDMD transgene sequence, the Assembly.fasta outputs were mapped to the custom GRCm38 + DMD + pYAC4 genome described above with minimap2 and assembled into super contigs using RagTag v 2.1.0 [34]. The contigs from the Assembly.fasta files were also converted into a BLAST v 2.14.1 + library that was queried with 500 bp segments from the 5′ and 3′ limits of the DMD locus in the hDMDTgSc transgene. Assembly graphs were viewed using Bandage v 0.8.1.

Transgene copy number determination

qPCRs were performed to quantify the hDMD transgene copy number and were done in technical triplicate. Each reaction well was set up using SYBR™ Green PCR Master Mix (Applied Biosystems), 50 nM of each forward and reverse primer and 10 ng gDNA in a 15 μL reaction volume. qPCR data was normalised to endogenous Sox1. Reactions were run on the QuantStudio™ 3 Real-Time PCR System (Applied Biosystems) using the Fast Run Mode with the following reaction conditions: 95 °C for 20 s, followed by 40 cycles of 95 °C for 1 s and 60 °C for 20 s, and a dissociation protocol to obtain a melt curve for all samples running from 60 to 95 °C.

RNA extraction and qRT-PCR

Muscle tissues were bead homogenised in 500 µL TRIzol reagent (Invitrogen) using Lysing Matrix D (MP Biomedicals). One hundred microliters of chloroform was added and samples were shaken vigorously, incubated at RT for 3 min then centrifuged at 10,000 g for 18 min at 4 °C to separate phases. The aqueous phase was isolated, mixed with an equal volume of ethanol and spun through a RNeasy column from the RNeasy Mini kit (Qiagen) at 8000 g for 30 s. The column was washed with 700 µL buffer RW1 and twice with 500 µL buffer RPE. RNA was eluted in 30 µL of nuclease-free water, quantified by nanodrop and stored at − 80 °C. cDNA was generated from 1 µg RNA using the High-Capacity RNA-to-cDNA kit (Applied Biosystems) as per manufacturer’s protocol.

qRT-PCRs were performed to quantify dystrophin transcript expression in technical and biological triplicate. The primers used were designed to target shared sequences in both hDMD and mDmd transcript. Each reaction well was set up using SYBR™ Green PCR Master Mix (Applied Biosystems), 50 nM of each forward and reverse primer and 1 µL of cDNA in a 15 μL reaction volume. qPCR data was normalised to beta-actin (Actb) expression. Reactions were run on the QuantStudio™ 3 Real-Time PCR System (Applied Biosystems) with the same parameters listed in the transgene copy-number determination section above. Analysis was performed using the Design and Analysis Software 2.7.0.

Western blot

Tissues were bead homogenised in disruption buffer (75 mM Tris HCl pH 6.8, 15% SDS, 1 × cOmplete Protease Inhibitor Cocktail EDTA (Roche)), using Lysing Matrix D (MP Biomedicals). 1/10 diluted lysates were quantified using the Pierce BCA protein assay (Thermo Scientific). Thirty micrograms of protein lysates were mixed with equal parts 2 × Loading buffer (21% w/v glycerol, 0.001% w/v bromophenol blue, 5% β-Mercaptoethanol), boiled at 95 °C for 5 min and spun at 21,300 rcf for 5 min prior to loading in NuPAGE Tris–Acetate gels (Invitrogen). Gels were run at 150 V for 1 h and transferred onto 0.2-µm PVDF membranes using the High MW setting on the Trans-Blot Turbo (Bio-Rad). Membranes were blocked with 10% skim milk in TBS-T for 2 h with gentle agitation at RT. Dystrophin was probed with 1:500 Anti-Dystrophin antibody, clone 2C6 MANDYS106 (Sigma-Aldrich) in 2% skim milk in TBS-T overnight at 4 °C. The following day, membranes were washed three times for 5 min with TBS-T and incubated with 1:5000 secondary anti-mouse antibody in 2% skim milk in TBS-T for 1 h at RT. After another set of TBS-T washes, membranes were developed with SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo Scientific), and chemiluminescent detection was performed on the ChemiDoc MP system (Bio-Rad) using automated exposure settings for intense bands. Membranes were stripped with Restore PLUS Western Blot Stripping Buffer (Thermo Scientific) prior to re-probing. All dystrophin was probed with 1:500 anti-dystrophin antibody MANDYS8. Vinculin was probed with 1:1 000 Anti-vinculin antibody, V9131 (Sigma-Aldrich).

Immunofluorescence

Fresh muscle was snap-frozen in dry-ice-cooled isopentane and immediately embedded in Tissue-Tek O.C.T. Ten-micrometre-thick cryostat-sliced transverse sections were permeabilised with PBS + 0.3% Triton X-100 for 10 min, blocked with 10% FCS in PBS + 0.3% Triton X-100 for 1 h at RT, and treated with 1 × ReadyProbes™ Mouse-on-Mouse IgG Blocking Solution (Invitrogen) as per manufacturer instructions. Slides were incubated with 1:100 Anti-Dystrophin antibody, clone 2C6 MANDYS106 (Sigma-Aldrich) diluted in blocking solution overnight at 4 °C in a humidified chamber. The next day slides were washed with PBS for 3 × 5 min and incubated with secondary anti-mouse antibody conjugated with Alexa Fluor® 594 for 1 h at RT. Slides were washed with PBS for 3 × 5 min and mounted with ProLong™ Gold Antifade Mountant with DAPI (Invitrogen) and coverslip. Images were taken on a Nikon Eclipse Ti2 microscope with a DS-Qi2 camera. The same image processing was applied to images from the same muscle group.

Histological analysis of muscles

Fresh muscle was mounted on 10% gum tragacanth on a piece of cork, snap frozen in liquid-nitrogen-cooled isopentane and immediately stored at − 80 °C. For skeletal muscles, 10-μm-thick cryostat-sliced transverse sections were stained with Lillie Mayer’s Haematoxylin and Eosin, as described by Wang et al. (2017) with the following modifications to the dehydration steps: 2 dips of 70% ethanol, 4 dips of 95% ethanol, 2 × 1 min of 100 ethanol and 2 × 1 min of xylene. For heart muscles, 10-μm-thick cryostat-sliced transverse sections were stained with Lillie Mayer’s Haematoxylin for 6 min rinsed with running tap water for 1 min, destained with 0.5% acid alcohol and developed in Scott’s tap water for 1 min [35]. After rinsing with running water for 2 min, slides were stained with eosin for 3 min and quickly rinsed with running tap water before dehydrating through an increasing concentration of ethanol and xylene as described in the skeletal muscle protocol. Slides were mounted with DPX mountant (Sigma-Aldrich) and coverslip. Images were taken using the NanoZoomer 2.0-HT at × 40 magnification.

Forelimb grip strength test

Muscle strength measurement was performed by a forelimb grip strength behaviour task [36]. Mice were tested at p32 and p60 using a grid attached to a Centor Easy 10N (Andilog) force gauge. Mice were suspended by the tail and allowed to grasp the grid tightly with both front paws, then pulled along a straight horizontal line away from the force gauge until their grip on the grid was broken. Measurements were repeated 4 times per set, and 3 sets were performed with a 1-min rest interval between sets. The highest measured rep was recorded for each set and normalised against body weight. Max force reported is the highest measured rep among all sets, and mean force reported is the average of the highest measured reps for each of all 3 sets.

Serum creatine kinase measurement

Thirty min after grip strength testing, Emla 5% local anaesthetic cream was applied to the mice’s tails, and they were warmed in a Mini Thermacage (datesand) for 10 min at 36 °C to dilate the tail vein. Blood samples were collected in microvettes from a nick to the lateral tail vein. The blood was allowed to clot at room temperature for 30 min, and the clot was separated from the serum by centrifuging at 2000 g for 10 min at 4 °C and immediately stored at − 80 °C. Mouse serum creatine kinase (CK) levels were measured by Gribbles Veterinary Pathology using the ADVIA Creatine Kinase CK_L assay on the ADVIA 1800 Chemistry System.