Abstract
Infantile-onset Pompe Disease (IOPD), caused by mutations in lysosomal acid alpha-glucosidase (Gaa), manifests rapidly progressive fatal cardiac and skeletal myopathy incompletely attenuated by synthetic GAA intravenous infusions. The currently available murine model does not fully simulate human IOPD, displaying skeletal myopathy with late-onset hypertrophic cardiomyopathy. Bearing a Cre-LoxP induced exonic disruption of the murine Gaa gene, this model is also not amenable to genome-editing based therapeutic approaches. We report the early onset of severe hypertrophic cardiomyopathy in a novel murine IOPD model generated utilizing CRISPR-Cas9 homology-directed recombination to harbor the orthologous Gaa mutation c.1826dupA (p.Y609*), which causes human IOPD. We demonstrate the dual sgRNA approach with a single-stranded oligonucleotide donor is highly specific for the Gaac.1826 locus without genomic off-target effects or rearrangements. Cardiac and skeletal muscle were deficient in Gaa mRNA and enzymatic activity and accumulated high levels of glycogen. The mice demonstrated skeletal muscle weakness but did not experience early mortality. Altogether, these results demonstrate that the CRISPR-Cas9 generated Gaac.1826dupA murine model recapitulates hypertrophic cardiomyopathy and skeletal muscle weakness of human IOPD, indicating its utility for evaluation of novel therapeutics.
Introduction
Generation of transgenic murine knock-in models of human disease once relied solely upon targeted insertion of the desired sequence via Cre-Lox recombination and embryonic stem cell implantation. Though this strategy has resulted in many successful murine model systems, it is labor-intensive, time-consuming, and expensive. The advent of genome editing via engineered nucleases, especially clustered regularly interspaced short palindromic repeats (CRISPR)-based systems has allowed for a potentially accurate, efficient, and relatively inexpensive alternative to the traditional method of transgenic knock-in model generation1,2.
As an intriguing example of disorders that may uniquely benefit from genome editing, inherited metabolic disorders (IMDs) are a diverse group of genetic diseases affecting the proper breakdown or synthesis of essential compounds such as carbohydrates, amino acids, or organic acids. Many of these disorders are caused by single gene defects that alter the expression and/or activity of critical metabolic enzymes. Given the monogenic nature of IMD pathogenesis, this class of genetic disorders is quickly becoming an area of high interest for CRISPR-mediated genome editing therapeutics3,4. Pompe disease, caused by the deficiency of acid α-glucosidase (GAA; EC 3.2.1.20), is characterized by lysosomal accumulation of glycogen in body tissues, primarily cardiac and skeletal muscle. Muscle lysosomal glycogen storage results in muscle weakness varying in age of onset and severity according to residual GAA enzymatic activity.
Infantile-onset Pompe disease (IOPD), caused by nearly absent GAA enzyme, typically manifests in the first two months of life with progressive and severe hypertrophic cardiomyopathy, heart failure, hypotonia, respiratory failure, and death within the first 14 months of life5.
Pompe disease can be treated with intravenous enzyme replacement therapy (ERT) using recombinant human acid α-glucosidase (rhGAA) enzyme, which significantly reduces cardiac hypertrophy and increases overall and ventilator-free survival6. Unable to endogenously synthesize GAA, Pompe patients are infused indefinitely and may produce an anti-rhGAA antibody response that may limit or neutralize treatment efficacy. Regardless of immune response, glycogen storage, autophagic buildup, and fibrosis within skeletal myocytes are observed even in early-treated IOPD patients7,8. Consequently, a phenotype of sensorineural hearing loss, central nervous system white matter abnormalities, slowly progressive muscle weakness and delayed mortality is now observed in rhGAA-treated survivors with IOPD9,10.
The limitations of current Pompe disease treatment underscore the necessity of new therapeutic development. CRISPR-based therapeutic strategies may address the impermanence of ERT, effecting permanent, highly-specific somatic correction of genomic Gaa mutations within myocytes and subsequent intramuscular, endogenous synthesis of enzyme. Reduced GAA enzyme within the bloodstream may also mitigate the immunogenicity of intravenous ERT.
First, though, an animal model with molecular, biochemical, physiological and functional analogy to human Pompe disease must be developed. Currently, there is a widely-utilized knockout murine model of Pompe disease (B6;129-Gaatm1Rabn/J) that demonstrates survival into adulthood with muscle glycogen storage and progressive muscle weakness11. This model bears a neomycin resistance cassette (~800bp) in its Gaa gene, not an analog of a human Gaa mutation, complicating preliminary efforts at in vivo genome correction. We report the successful generation of a Gaac.1826dupA (p.Y609*)12 murine knock-in model of Pompe disease utilizing a novel, dual-single guide RNA approach flanking the intended Gaa insertion site, and early characterization that demonstrates GAA enzymatic deficiency, hypertrophic cardiomyopathy, muscular glycogen storage and pathology recapitulating human Pompe disease.
Results
Dual overlapping gRNA approach achieves highest HDR levels in vitro
Three guide RNAs (Gaac.1826 gRNA-1,2,3) were selected based on best predicted on-target and off-target scoring and proximity of expected cut site to Gaac.1826 target locus (Figure 1A). To determine in vitro on-target editing activity and HDR efficiency, Gaac.1826 gRNA expression vectors and respective ssODN donor templates were nucleofected into C2C12 mouse myoblasts. On-target editing activity and HDR efficiency was highly dependent on target sequence (Table 1). For single gRNA approach, Gaac.1826 gRNA-3 demonstrated the highest on-target editing activity (47.9±0.1%) with Gaac.1826 gRNA-2 achieving the best HDR efficiency (7.7±1.4%). Given that multiple gRNAs with overlapping sequences are known to enhance CRISPR/Cas9-mediated knock-in efficiency24, we evaluated whether a dual overlapping gRNA approach could improve our on-target editing activity and HDR efficiency. We chose to test Gaac.1826 gRNAs-2 and −3 at an equimolar ratio as they achieved the highest levels of on-target editing activity and HDR efficiency. Furthermore, Gaac.1826 gRNAs-2 and −3 are a sense-antisense pair with fully overlapping target sequences thereby reducing the likelihood of added off-target activity. We found that the dual overlapping gRNA approach achieved high on-target editing activity (47.9±0.1%) with the highest overall HDR efficiency (12.6±2.9%). Further testing will need to be performed to confirm that this dual overlapping gRNA approach can be broadly applied to increase HDR efficiency in other target loci and cell lines.
Generation & characterization of Gaac.1826dupA knock-in C2C12 cell line
We used the dual overlapping Gaac.1826 gRNA strategy (Figure 2A,B) followed by puromycin-resistant selection to isolate Gaac.1826dupA knock-in C2C12 clonal cells. Sanger sequencing results confirmed presence of desired Gaac.1826dupA knock-in mutation as well as silent PAM and seed region mutations to prevent gRNA editing of the donor template (Figure 1B). Gaac.1826dupA knock-in cells exhibited enhanced PAS staining – a marker of glycogen accumulation – relative to Gaawt cells (Figure 1C). Gaac.1826dupA knock-in cells display a 96% reduction in Gaa transcript levels and GAA enzymatic activity was completely abolished (Figure 1D). Together, these results demonstrate that the dual overlapping gRNA approach can increase overall HDR efficiency in vitro thus improving the probability of isolating clonal cells with a desired knock-in mutation. Moreover, our Gaac.1826dupA knock-in cell line exhibits molecular and biochemical analogy to human Pompe disease thereby validating its use as an in vitro model.
Generation & characterization of Gaac.1826dupA transgenic mice
We next applied the dual overlapping Gaac.1826 gRNA strategy in vivo (Figure 2A,B) to generate transgenic Gaac.1826dupA knock-in mice via pronuclear injection of C57BL/6NJ single-cell embryos by standard methods17. The dual overlapping Gaac.1826 gRNA strategy achieved 66.7% on-target editing activity (founder mice positive for any Gaa mutation) and 25% HDR efficiency (founder mice positive for Gaa c.1826dupA mutation) (Table 2). Following founder mice genotyping, we selected a founder with the lowest levels of mosaicism-as determined by TIDER16 analysis – for mating and segregation of Gaa c.1826dupA mutation. We successfully generated a homozygous Gaa c.1826dupA knock-in mouse after 2 generations of breeding (Figure 2C). Sequencing results confirmed presence of desired Gaac.1826dupA knock-in mutation as well as silent PAM and seed region mutations in G0 founder (GaaMosaic), G1 heterozygous (Gaawt/c.1826dupA) and G2 homozygous knock-in (Gaac.1826dupA) mice (Figure 2D).
To determine the extent of genomic mosaicism in our transgenic mice, we performed whole genome sequencing at >50x coverage of the G0 founder (GaaMosaic), G1 heterozygous (Gaawt/c.1826dupA) and G0 wild-type (Gaawt) mice. On-target locus alignment of GaaMosaic to Gaawt demonstrates 63% Gaac.1826dupA knock-in mutation (32/51 reads) and 37% indels (19/51 reads). Alignment of Gaawt/c.1826dupA to Gaawt resulted in 33% Gaac.1826dupA knock-in mutation (17/51 reads), 63% no mutation (32/51 reads) and 4% nonspecific mutation (2/51 reads) (Figure 2E). Together, these results demonstrate that the desired Gaac.1826dupA knock-in mutation efficiently integrated into our founder mouse genome and was successfully passed onto its progeny.
To screen for off-target integration of the donor template, we searched for called single nucleotide variants (SNVs) that had the unique donor template motif. We then repeated this step for the reverse complement and found that the only positive result was the intended mutation at the Gaac.1826 target locus. Next, we screened the top 5 genomic regions predicted by GT-Scan14 to be potential off-target sites (Figure 2F). There were no detected SNVs within 500bp of these sites.
Altogether, these results suggest that the dual overlapping gRNA approach is an efficient strategy to generate transgenic Gaac.1826dupA knock-in mice and did not result in any detectable off-target activity in the genomes of our founder mouse and its progeny.
Gaa transcript levels, GAA enzyme activity and glycogen load in Gaac.1826dupA transgenic mice
Knock-in of the Gaac.1826dupA mutation leads to a frameshift in the Gaa coding sequence resulting in a premature stop codon at amino acid position 609 (p.Tyr609*). Nonsense mediated decay of transcripts containing premature stop codons prevents expression of truncated and potentially deleterious proteins. Thus, we assessed nonsense mediated decay by measuring Gaa transcript levels from Gaac.1826dupA transgenic and wild-type mouse tail samples using quantitative real-time PCR (qRT-PCR). Transgenic mice demonstrated a gene dose-dependent decrease in Gaa transcript levels relative to Gaawt (WT) mice (Figure 3A). Gaac.1826dupA homozygous (KI) and Gaawt/c.1826dupA heterozygous (HET) mice exhibited a 75% and 46% reduction in Gaa transcript levels relative to WT, respectively. To compare these results with the commercially available Gaatm1Rabn knockout (KORabn) mice, we found an 81% reduction in KORabn Gaa transcript levels relative to WT. Notably, there was no significant difference in Gaa transcript levels between KI and KORabn mice.
Next, we measured GAA enzyme activity levels in Gaac.1826dupA transgenic and WT liver, diaphragm and gastrocnemius muscle. For all tissues, transgenic mice demonstrated a gene dose-dependent decrease in GAA enzyme activity levels relative to WT mice (Figure 3B). Liver GAA activity was reduced by 100% and 35% reduction in KI and HET mice relative to WT, respectively. Diaphragm GAA activity was reduced by 95% and 54% in KI and HET mice relative to WT, respectively. Gastrocnemius muscle GAA activity was reduced by 98% and 57% in KI and HET mice relative to WT, respectively. We also assessed GAA activity in KORabn mice and found a 99%, 88% and 97% reduction in liver, diaphragm and gastrocnemius GAA activity relative to WT, respectively. Notably, there was no significant difference in GAA enzymatic activity between KI and KORabn mice for all tissue types.
We then measured glycogen load in Gaac.1826dupA transgenic and WT diaphragm and gastrocnemius muscle. For both tissues, transgenic mice demonstrated a gene dose-dependent increase in glycogen load relative to WT mice (Figure 3C). Diaphragm glycogen levels were increased 108-fold and 2-fold in KI and HET mice relative to WT, respectively. Gastrocnemius muscle glycogen levels were increased 28-fold and 2-fold in KI and HET mice relative to WT, respectively. We also measured glycogen load in KORabn mice and found an 82-fold and 89-fold increase in diaphragm and gastrocnemius glycogen levels relative to WT, respectively. Interestingly when compared to KI, KORabn glycogen load was 1.3-fold lower and 3.2-fold higher in diaphragm and gastrocnemius, respectively.
Taken together, these results demonstrate that our Gaac.1826dupA knock-in mouse model exhibits molecular and biochemical analogy to the established preclinical model of Pompe disease (Gaatm1Rabn) and is an appropriate in vivo model for genome-based therapeutic evaluation.
Cardiac anatomy and function in Gaac.1826dupA transgenic mice
To assess overall cardiac anatomy and function in Gaac.1826dupA transgenic mice, we performed echocardiography on 3-month old WT, HET and KI mice (Figure 4A). Relative to WT and HET mice, KI mice display significant increases in intraventricular septal diameter (IVSd), left ventricular posterior wall diameter (LVPWd) and left ventricle mass index – anatomical hallmarks of hypertrophic cardiomyopathy (Figure 4B). KI mice also display alterations in left ventricular internal diameter end systole (decreased; relative to HET) and fractional shortening (increased; relative to WT) – early indicators of abnormal cardiac function (Supp. Figure 1).
These results demonstrate that Gaac.1826dupA knock-in mice exhibit early-onset hypertrophic cardiomyopathy – a primary clinical feature of IOPD. Moreover, this work displays the utility of murine echocardiography as a robust diagnostic tool to assess cardiomyopathy in preclinical disease models.
Forelimb grip strength performance in Gaac.1826dupA transgenic mice
To assess forelimb muscle strength in Gaac.1826dupA transgenic mice, we measured peak tension force exerted by 3-month old WT, HET and KI mice using a murine-specific grip strength meter. Both male and female KI mice exhibited significant decreases in peak tension force when compared to WT and HET mice as well as a significant reduction in body mass relative to WT mice (Figure 5).
These results show that Gaac.1826dupA knock-in mice exhibit early-onset musculoskeletal impairment – a key feature of IOPD. Gaac.1826dupA knock-in mice also display a reduction in overall body mass which may be an early indicator of muscular dystrophy.
Gaac.1826dupA transgenic mouse histology
To examine cardiac and skeletal muscle tissue structure and glycogen load, we performed periodic acid-Schiff (PAS) staining of heart, diaphragm and gastrocnemius muscle from 3-month old WT and KI mice. In contrast to WT mice, KI mice displayed aggregation of PAS staining in all three tissue types (Figure 6). Furthermore, when compared to WT muscle fibers, KI cardiac and skeletal muscle fibers appear irregular in shape and display abnormal extracellular spacing between cells. Altogether, these results show that Gaac.1826dupA knock-in mice display early signs of muscle tissue pathology. The observed PAS-positive aggregates and irregular myocyte structural features are key markers of disease pathogenesis and progression in Pompe tissue.
Discussion
Currently, preclinical development of novel therapeutic options for Pompe disease rely primarily on the Gaatm1Rabn/J knockout mouse model, developed in the late 1990s. While the Gaatm1Rabn/J mouse is an appropriate model for evaluating new enzyme replacement and gene therapy strategies, a Pompe disease knock-in model bearing a Gaa mutation homologous to a known human pathogenic variant is much preferred for development of genome correction-based therapeutics.
This study demonstrates the successful generation of a new knock-in model of Pompe disease using CRISPR-Cas9 genome editing. Our data show the importance of optimizing HDR-mediated knock-in efficiency via in vitro gRNA and donor template testing prior to in vivo application. We found that in silico combined rank scoring of gRNAs does not always correlate with actual experimental results. In fact, our Gaac.1826 gRNA with the highest predicted rank score (gRNA-1) demonstrated the lowest in vitro on-target efficacy. Our data also suggest the enhancement of both gene editing and HDR events by utilizing a dual/multiple gRNA approach versus a single gRNA approach24, especially if there is overlap of candidate gRNA target sequences. We found that using a dual gRNA approach, with complete overlap in gRNA target sequences, resulted in the highest level of knock-in efficiency (12.6%) when compared to each gRNA alone (0 to 7.7%). and higher than the additive knock-in efficiencies of the gRNAs (11.2%).
Our preliminary in vitro results provide empirical evidence that the dual gRNA approach could increase the probability of generating cellular and murine knock-in disease models. Consequently, we successfully isolated and characterized a clonal murine C2C12 myoblast line bearing a known pathogenic Pompe disease mutation - Gaac.1826dupA – using the dual gRNA strategy. We found that Gaac.1826dupA knock-in cells display molecular and biochemical analogy to human Pompe disease with significantly reduced Gaa transcript levels, undetectable GAA enzymatic activity and increased glycogen load. Further testing will need to be performed to confirm that this dual overlapping gRNA approach can be broadly applied to increase knock-in efficiency in other target gene loci and cell lines.
In vivo application of the dual gRNA approach via pronuclear microinjection of fertilized zygotes resulted in the successful generation of a Gaac.1826dupA knock-in mouse model which exhibits molecular, biochemical, physiologic and histologic homology to human Pompe disease. Our Gaac.1826dupA whole genome sequencing results support a recent study25 confirming high on-target and undetectable off-target activity in the edited genomes of transgenic mice. Like the Gaatm1Rabn/J knockout model9, Gaac.1826dupA knock-in mice display reduced Gaa mRNA transcript levels, undetectable GAA enzymatic activity, and increased glycogen load in multiple tissue types. This disruption in gene expression, enzymatic activity and substrate clearance in Gaac.1826dupA knock-in mice manifests as early-onset cardiac hypertrophy and impaired skeletal muscle function - key phenotypic hallmarks of IOPD5. Gaac.1826dupA knock-in mice also display critical histopathological features of Pompe disease including glycogen accumulation in cardiac and skeletal muscle tissue. Interestingly, the Gaac.1826dupA knock-in mice do not exhibit a lethal phenotype despite development of hypertrophic cardiomyopathy at age 3 months. Long-term phenotyping of Gaac.1826dupA knock-in mice will provide further evidence of analogy to human Pompe disease. We are currently performing longitudinal physiological and histological assessment of Gaac.1826dupA knock-in mice to determine natural history and disease progression. Given the importance of determining cross-reactive immunogenic material (CRIM) status as it relates to severity of disease progression and immune response to GAA enzyme replacement therapy26, we will also aim to determine immune response of Gaac.1826dupA knock-in mice to recombinant GAA protein. Altogether, this study provides evidence that Gaac.1826dupA knock-in C2C12 myoblast cells and mice recapitulate, with the exception of infantile mortality, IOPD. Our results validate their use as models of Pompe disease for preclinical evaluation of genome correction-based and other potential therapeutic strategies.
Materials & Methods
Gaac.1826 target locus guide RNA and donor ssODN design
In silico design of CRISPR-Cas9 guide RNAs (gRNAs) specific for the Gaac.1826 target locus was performed using Genetic Perturbation Platform (GPP) sgRNA Designer13 (Broad Institute). Candidate gRNAs were selected using the following criteria: 1) top combined rank score (based upon on-target efficacy and off-target specificity scores) and 2) proximity of predicted Cas9 nuclease cut site to the Gaac.1826 target locus.
Further potential gRNA off-target analysis was performed using Genome Target Scan (GT-Scan)14. Three gRNAs were used in this study (Table 1): gRNA-1 (5’-ACGGCCGGTACGCTGGTCAC-3’), gRNA-2 (5’-GTACGCTGGTCACTGGACAG-3’), gRNA-3 (3’-CTGTCCAGTGACCAGCGTAC-5’).
All donor single stranded oligodeoxynucleotides (ssODNs) were designed with 50bp homology arms flanking the target locus and silent mutations in the protospacer adjacent motif (PAM) and seed region (5nt upstream of PAM) and synthesized by Integrated DNA Technologies.
Gaac1826 guide RNA spCas9 expression vector cloning
gRNA oligonucleotides with BbsI restriction enzyme overhangs were designed as follows: Forward oligo (5’-CACCG(gRNA)-3’); Reverse oligo (5’-AAAC(reverse complement gRNA)C-3’). Complementary gRNA oligonucleotides were annealed and ligated to BbsI-digested pSpCas9(BB)-2A-Puro plasmid (Addgene plasmid ID# 48139) using Quick Ligation Kit (New England Biolabs). Positive pSpCas9(BB)-gRNA clones were confirmed by Sanger sequencing and expanded using PureLink® HiPure Plasmid Midiprep Kit (Invitrogen).
In vitro testing of Gaac1826 guide RNAs
pSpCas9(BB)-2A-Puro-Gaac.1826 gRNA expression vectors and donor ssODNs were transfected into C2C12 murine myoblast cells (ATCC® CRL-1772TM) using nucleofection-based transfection (Neon® transfection system, Invitrogen). For nucleofection-based transfection, 3×105 cells were resuspended in a reaction mixture containing 4.5μg pSpCas9(BB)-2A-Puro-Gaac.1826 gRNA expression vector(s) and 450nM ssODN. Cells were electroporated using the following parameters - Pulse voltage: 1650V, Pulse width: 10ms, Pulse number: 3 – and plated onto duplicate wells of Matrigel®-coated 6-well culture plates containing 2mL culture media (DMEM + 10% FBS, 2mM GlutaMax, 100U/mL penicillin, 100μg/mL streptomycin, 0.25μg/mL amphotericin B) and maintained at 37°C with 5%CO2. 48h post-transfection, cellular genomic DNA was extracted using QuickExtract DNA solution (Epicentre) and the Gaac.1826 target locus was PCR amplified and purified using DNA Clean & ConcentratorTM (Zymo Research) and Sanger sequencing was performed (Retrogen). spCas9 nuclease activity and homology-directed repair (HDR) knock-in efficiency were determined by Tracking of Indels by Decomposition (TIDE)15 or Tracking of Insertion, Deletions, and Recombination events (TIDER)16 analysis of DNA sequence electropherogram files.
Generation of Gaac.1826dupA knock-in C2C12 cell line
4.5μg of pSpCas9(BB)-2A-Puro-Gaac.1826 gRNA-2 and pSpCas9(BB)-2A-Puro-Gaac.1826 gRNA-3 expression vectors (1:1 molar ratio) and 450nM ssODN were added to 3×105 cells and electroporated with similar parameters described above. pCMV6-AC-GFP (OriGene) was used as a transfection positive control and puromycin resistance gene negative control. Nucleofected cells were plated onto duplicate wells of Matrigel®-coated 6-well culture plates for 24 h before adding 2.5μg/mL puromycin dihydrochloride (Sigma-Aldrich) for selection. Puromycin-containing media was replaced every 48h until all pCMV6-AC-GFP transfected cells were no longer viable. Following puromycin-resistant selection, single cell clones were selected by standard serial dilution methods in 96-well plates in the presence of 2.5μg/mL puromycin dihydrochloride. Single cells clones were identified and maintained until sequencing results confirm clonal cell genotype.
Generation of Gaac.1826dupA knock-in mice
All study procedures were reviewed and approved under University of California Irvine’s IACUC protocol #AUP 16-63. Pronuclear stage C57BL/6NJ embryos were produced by standard methods17. In brief, gRNAs were designed using Genetic Perturbation Platform (GPP) sgRNA Designer13 and off targets were analyzed by GTScan14. 3μM crRNA/tracrRNA/3xNLS-Cas9 protein and 10ng/μL ssODN (IDT) were injected into pronuclei. Surviving embryos were implanted into oviducts of 0.5dpc pseudopregnant ICR females.
Experimental Animals
The use and care of animals used in this study adhere to the guidelines of the NIH Guide for the Care and Use of Laboratory Animals, which are utilized by the CHOC Children’s Institutional Animal Care and Use Committee. All study procedures were reviewed and approved under CHOC Children’s IACUC protocol #160902.
Heterozygous (Gaawt/c.1826dupA) males and females were crossed to obtain homozygous knock-in (KI), heterozygote (HET) and wild type (WT) mice for this study. Experiments were performed on age- and sex-matched mice. Genotyping was confirmed by Sanger sequencing of the Gaac.1826 target locus.
Quantitative real-time PCR
Total RNA was extracted from C2C12 myoblasts or postnatal day 21 tail tip samples using Direct-zol RNA miniprep kit (Zymo Research) and reverse-transcribed using High Capacity cDNA Reverse Transcription kit (Applied Biosystems) following manufacturer’s instructions. TaqMan® Fast Advanced master mix (Applied Biosystems) and specific TaqMan® primer/probe assays for Gaac.1826wt: Forward primer 5’-GGAACACGACCCTTTGTGAT-3’; FAM-hybridized probe 5’ GTACGCTGGTCACTGGACAG-3’; Reverse primer 3’-ATGCAAGATGCTCCCAAGAG-5’ or Gapdh (TaqMan® assay #Mm99999915_g1) were added to cDNA samples and amplified in triplicate. Gapdh was used as an internal reference gene, and relative quantification of Gaa gene expression was measured by the comparative ΔCt method.
GAA enzymatic activity assay
For biochemical analysis, frozen C2C12 myoblast pellets or mouse tissues were homogenized in CelLytic M cell lysis reagent (MilliporeSigma). α-glucosaidase enzyme activity was assessed as previously described with minor modifications22. In brief, 10 µL tissue homogenate was mixed with 10 µL of 6 mM 4-methylumbelliferyl-α-D-glucopyranoside substrate (4-MUA, MilliporeSigma) in McIlvaine citrate/phosphate buffer in pH 4.5 and quenched with 180 µL glycine carbonate buffer, pH 10.5 after 1 hr incubation at 37°C in a 96-well plate format. Fluorescence measurements were obtained using an FLx800 spectrofluorophotometer (BioTek) at excitation and emission wavelengths of 360 nm and 460 nm, respectively. In order to eliminate the background activity caused by maltase-glucoamylase in liver tissues, a final concentration of 3 µM acarbose (Cayman Chemical Company) was added in α-glucosaidase enzyme activity reaction for liver samples. One activity unit was defined as 1 nmol converted substrate per hour. Protein concentration was estimated using Pierce BCA assay kit and bovine serum albumin was used as a standard. Specific activity was defined as units of activity per mg of protein.
Glycogen assay
Tissue glycogen levels were measured using a glycogen assay kit (Sigma-Aldrich) following manufacturer’s instructions. In brief, 10 µL tissue homogenate was incubated with hydrolysis enzyme reaction mixture in a total volume of 50 µL at room temperature for 30 min before adding 50 µL development enzyme reaction mixture for 30 min incubation at room temperature. Absorbance at 570 nm was measured using a spectrophotometer (Multiskan FC Microplate Photometer, Thermo Fisher). A standard curve was generated using standard glycogen solution in the reaction. A reaction without hydrolysis enzyme treatment was used for background correction (endogenous glucose) for each sample.
Periodic acid-Schiff staining
C2C12 cell lines were seeded on Matrigel®-coated 18mm glass coverslips at low density (2.5×103 cells) in culture media at 37°C with 5%CO2. 24h post-plating, culture media was replaced with serum-free culture media and maintained for an additional 72 hours with daily replacement of serum-free culture media. 96h post-plating, cells were fixed with 4% paraformaldehyde (4% PFA, Electron Microscopy Sciences) for 30 min at room temperature. Fixed cells were periodic acid-Schiff (PAS) stained (purple-magenta) and hematoxylin counterstained (dark blue) per manufacturer’s protocol (Sigma) and mounted on glass slides with ProLong Gold antifade mountant (ThermoFisher). Representative images were captured on a bright-field microscope (Olympus), at 40x/0.55NA objective magnification.
Whole genome sequencing and analysis
Whole genome sequencing and analyses were performed on G0 founder (GaaMosaic) and G1 heterozygous (Gaawt/c.1826dupA) tail samples. In brief, 1 μg fragmented genomic DNA was ligated with adaptors using TruSeq DNA libraries and whole genome sequencing was performed on an Illumina HiSeq X Ten Sequencer at 40-50x read depth (Fulgent Genetics). WGS on-target and on-and off-target analysis was analyzed on OnRamp BioInformatics platform. Data were aligned to the Mouse genome (mm10) using BWA18. PCR artifacts were identified with the memtest utility from Sentieon19, and filtered out using samtools20. Alignments were de-duplicated and realigned around insertions and deletions using LocusCollector, Dedup and Realigner from Sentieon. SNV variant calling was performed with GVCFtyper from Sentieon, using the mouse dbSNP 142 data (http://hgdownload.cse.ucsc.edu/goldenpath/mm10/database/snp142.txt.gz) as the known SNPs. Known SNPs and variants falling in un-located chromosomes were removed from the analysis.
For on-target analysis, Gaac.1826 target loci from aligned FASTQ reads were designated to the following event categories: 1) Gaac.1826dupA knock-in mutation, 2) insertion/deletion mutation (indel), 3) no mutation, 4) nonspecific mutation. Data are presented as stacked bar graphs indicating percentage of WGS on-target reads for each event category
For off-target analysis, we searched for called SNVs that had inserted A, and were flanked by a N->A mutation 4 bases upstream, a N->T mutation 17 bases downstream, and a N->A mutation 20 bases downstream, and then repeated this step for the reverse complement. The only result was the intended Gaac.1826dupA mutation. 5 genomic regions were predicted by GT-Scan14 to potential be off-target sites of the CRISPR sgRNAs. There were no detected SNVs within 500bp of these sites. The fully processed BAM files (after Realigner) were used as input to the Manta structural variant caller21. For each of the non-WT samples, we ran the Manta Somatic caller with the C57BL6-WT sample as ‘normal’ and the sample of interest as ‘tumor’, thereby subtracting the background structural variants in C57BL6-WT compared to mm10. We used vcf-annotate (https://vcftools.github.io) to annotate the output VCF files from Manta.
Murine echocardiography
Prior to echocardiography, a depilatory cream was applied to the anterior chest wall to remove the hair. 3-month old mice were anesthetized with 5% isoflurane for 15 seconds and then maintained at 0.5% throughout the echocardiography examination. Small needle electrodes for simultaneous electrocardiogram were inserted into one upper and one lower limb. Transthoracic echocardiography (M-mode and 2-dimensional echocardiography) was performed using the FUJIFILM VisualSonics Inc., Vevo 2100 high-resolution ultrasound system with a linear transducer of 32-55MHz. Measurements of chamber dimensions and wall thicknesses were performed. Percentage fractional shortening (%FS) is used as an indicator of left ventricular systolic cardiac function and is calculated as follows: %FS = LVIDd – LVIDs / LVIDd * 100.
Forelimb grip strength assay
Forelimb grip strength was measured as previously described23. One hour prior to grip strength measurement, 3-month old mice were transferred to behavioral room to acclimate subjects to test conditions. Following acclimatization, each mouse was weighed and placed on a forelimb pull bar. Peak tension force exerted by each animal was recorded by a mouse grip strength meter (Columbus Instruments). Each mouse performed 3 pulls per day over 3 consecutive days for a total of 9 pulls per test session. Peak tension force (N) was calculated as the average of each subject’s 9 pulls over the test session.
Tissue Harvesting, Processing, and Histological Staining
Tail biopsies for genotyping were collected on postnatal day 7 (founder mice) or postnatal day 21 (G1 & G2 mice). Genomic DNA was extracted using Agencourt® DNAdvanceTM genomic DNA isolation kit (Beckman Coulter) with proteinase K and DTT. 3-month old mice were euthanized using CO2 asphyxiation and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% PFA for histological staining or PBS alone for biochemical analyses. Heart, diaphragm and gastrocnemius muscle tissue were harvested in this study. Tissue samples for biochemical studies were rapidly frozen and stored at −80°C; tissues for histological staining were processed and embedded in paraffin blocks for sectioning at 4μm thickness and periodic acid-Schiff (PAS) staining was performed.
Statistical analysis
All graphs and statistical comparisons were generated using GraphPad Prism 8. Statistical analyses were performed using the two-tailed unpaired t-test or one-way ANOVA followed by Tukey’s HSD test. All data are presented as mean ± SD.
Funding
This work was supported by the Campbell Foundation of Caring (JYH, SHK, RYW) and the UCLA Intercampus Medical Genetics Training Program Institutional Training Grant Award #2T32GM008243-31 (JYH).
Author Contributions
JYH and RYW conceived and planned experiments. JYH, SHK, ADR, RYW, JN, NDD, YC, and JDT performed experimental procedures and critically reviewed the manuscript. JYH and JN designed the Cas9 sgRNAs. JYH designed the ssODN template and Gaa spCas9 transfection plasmids. JN performed the murine pronuclear microinjections and subsequent embryo implantations. JYH, RYW, SHK, ND, and JDT wrote the manuscript.
Competing Interests
JDT is an employee of OnRamp Bioinformatics. The remaining authors have no competing interests to declare.