CRISPR-mediated era and characterization of a Gaa homozygous c.1935C>A (p.D645E) Pompe illness knock-in mouse mannequin recapitulating human childish onset-Pompe illness

Gaa
^c.1935 goal locus information RNA and donor ssODN design

In silico design of CRISPR-Cas9 information RNAs (gRNAs) particular for the Gaa^c.1935 goal locus was initially carried out utilizing CRISPick, the Genetic Perturbation Platform (GPP) sgRNA Designer¹³. Candidate gRNAs have been chosen utilizing the next standards: 1) high mixed rank rating (primarily based on on-target efficacy and off-target specificity scores) and a couple of) proximity of predicted Cas9 nuclease reduce website to the Gaa^c.1935 goal locus. Additional potential gRNA off-target evaluation was carried out utilizing Genome Goal Scan (GT-Scan)¹⁴. Two gRNAs have been first chosen for use in producing Gaa^c.1935C>A KI C2C12 cells: gRNA-1 (5′- CGCAGATGTCCGCCCCGACC-3′), and gRNA-2 (5′- GCAGATGTCCGCCCCGACCA-3′).

Technology and characterization of Gaa
^c.1935C>A KI C2C12 cell line

Gaa^c.1935 gRNA-1 and gRNA-2 expression vectors and their respective single-stranded donor oligonucleotides (ssODN) have been electroporated into C2C12 mouse myoblasts to evaluate in vitro on-target modifying and HDR effectivity. Gaa^c.1935 gRNA-2 demonstrated larger on-target modifying (26.7 ± 10.7%) and HDR effectivity (5.4 ± 3.4%) than gRNA-1 (on-target modifying: 13.2 ± 3.7%; HDR effectivity: 3.8 ± 0.6%) (Desk 1). Following puromycin-resistant choice, we have been capable of efficiently isolate and increase Gaa^c.1935C>A KI C2C12 clonal cells electroporated with Gaa^c.1935 gRNA-1 and/or gRNA-2 and their respective donor ssODN (Desk 1; Fig. 1A). Sanger sequence outcomes confirmed that the Gaa^c.1935C>A KI mutation together with a Gaa^c.1920C>T silent protospacer adjoining motif (PAM) mutation have been efficiently launched into the clonal line (Fig. 1B).

Technology and characterization of a Gaa^c.1935C>A C2C12 myoblast clonal cell line. (A) Sequences of information RNAs concentrating on the Gaa^c.1935 goal locus. Horizontal arrow signifies antisense information RNAs used on this research. Protospacer adjoining motifs (PAM; NGG) are highlighted in coloration akin to the respective information RNA. The Gaa^c.1935 locus for focused cytosine to adenine transversion is highlighted in crimson. (B) Sanger sequencing chromatograms of controls (Gaa^wt) and clonal KI (Gaa^c.1935C>A) C2C12 myoblast genomic DNA on the Gaa^c.1935 locus. Black arrow signifies a synonymous mutation on the PAM website (Gaa^c.1920C>T). Crimson arrow signifies the specified KI mutation (Gaa^c.1935C>A). Grey shaded area signifies amino acid change from aspartic acid (Asp; GAC) to glutamic acid (Glu; GAA) at place 645. (C) Periodic-acid Schiff (PAS) staining of management (Gaa^wt) and clonal KI (Gaa^c.1935C>A) C2C12 myoblasts. Fastened cells have been stained by PAS staining (purple-magenta) and counterstained by hematoxylin (blue). Solely Gaa^c.1935C>A KI myoblasts show vital amassed PAS staining (see arrows). Consultant photos have been captured on a bright-field microscope at 20 × goal magnification. Scale bar represents 50 µm. (D) GAA enzymatic exercise in Gaa^wt and Gaa^c.1935C>A C2C12 myoblasts. Very low GAA exercise (~ 2.3% of wt) was measured in Gaa^c.1935C>A C2C12 myoblasts in comparison with Gaa^wt C2C12 myoblasts. GAA enzymatic exercise was measured utilizing a fluorometric 4-MU α-d-glycoside assay and normalized to whole quantity of pattern protein. Knowledge generated from three unbiased experiments are proven as imply ± SD. Comparisons have been analyzed with unpaired one-tailed t-tests. ****p < 0.0001.

Compared to Gaa^wt cells, Gaa^c.1935C>A KI cells displayed elevated PAS staining, indicating the buildup of glycogen (Fig. 1C). Moreover, GAA enzymatic exercise was virtually abolished in Gaa^c.1935C>A KI cells relative to Gaa^wt cells; lower than 2.3% of WT GAA exercise was detected within the KI cell line (Fig. 1D). Taken collectively, these outcomes reveal that our Gaa^c.1935C>A KI C2C12 cell line reveals a molecular and biochemical phenotype noticed in human PD and may be utilized as an in vitro mannequin for additional research.

Technology and characterization of Gaa
^em1935C>A transgenic mice

Given our prior success in producing Gaa^em1826dupA KI cell and transgenic mouse strains utilizing a bi-directional, twin overlapping gRNA technique¹⁵, an extra gRNA-3 (5′-GGGCGTGCCCCTGGTCGGGG-3′) was launched (Fig. 2A). Evaluating gRNA-3 with gRNA-1 by in silico evaluation, gRNA-3 had larger predicted on-target effectivity (0.5487 [gRNA-3] vs 0.3905 [gRNA-1]) by CRISPick¹³ in addition to decrease predicted off-targets by GT-Scan¹⁴. We then utilized the twin overlapping gRNA technique in vivo utilizing Gaa^c.1935 gRNA-2 and gRNA-3 with the ssODN (Fig. 2B) through pronuclear injection of C57BL/6NJ single-cell embryos by commonplace strategies¹⁶. 566 oocytes have been injected, 531 oocytes (94.8%) have been implanted, and a complete of 39 founder mice have been generated. The twin overlapping gRNA methodology achieved a excessive share of on-target modifying exercise in genome-edited founder mice (89.7%; 35 out of the 39 mutant mice) exhibiting vital Cas9 exercise/insertion/deletion (indel) mutations inside the goal area in Gaa. Among the many founders, 15 mutants (38.5%) exhibited on Sanger sequencing the specified c.1935C>A KI mutation (See Desk 2) together with the silent PAM and seed area mutations (Fig. 2C). Of those 15, 13 had vital (> 40% estimated by Sanger sequencing) indel mutations within the Gaa goal area. Founder #1 (> 50% for Gaa^c.1935C>A) and founder #2 (> 25% for Gaa^c.1935C>A) had the best share of the c.1935C>A mutation and lowest share of indels. The 2 founder mice and Gaa^wt mice underwent entire genome sequencing (WGS) at > 50 × protection and on-target locus alignment evaluation to higher quantitate the extent of genomic mosaicism. For on-target evaluation, Gaa^c.1935 goal loci from aligned FASTQ reads have been designated to 4 classes: Gaa^c.1935 KI mutation; indel mutation; no mutation; and nonspecific mutation. WGS evaluation demonstrated extremely environment friendly integration of the specified Gaa^c.1935C>A KI mutation with indel and nonspecific mutations comprising a minority of genomic modifying occasions in Gaa^c.1935C>A founder mice (Fig. 2D). For off-target evaluation of the WGS knowledge, first we examined the seven genomic areas (Supplementary Desk 1) predicted by GT-Scan as potential off-target websites of gRNA-2 and gRNA-3, and the one outcome was the supposed Gaa^c.1935C>A mutation. No single nucleotide variations (SNVs) have been detected inside 500 bp of those websites. Subsequent, we examined the founders’ WGS knowledge for any de novo (in comparison with WT WGS) C>A transversions with a de novo N > A mutation 3 or 6 bases upstream, an N>C mutation 12 bases upstream, or an N>T mutation 15 bases upstream of the C>A suggesting ectopic SpCas9/HDR exercise. No ectopic HDR signatures have been recognized within the genomes of both founder #1 or founder #2.

Technology of a Gaa^em1935C>A transgenic mouse line. (A) Twin overlapping information RNA method concentrating on the Gaa^c.1935 goal locus. Arrowhead route signifies whether or not information RNA is sense (proper) or antisense (left). PAM sequences (NGG) are highlighted in coloration akin to the information RNA arrows. The Gaa^c.1935 locus for goal adenine to cytosine nucelotide transversion is highlighted in crimson. Anticipated Cas9 nuclease reduce websites are proven as vertical arrows in coloration akin to the information RNA arrows. (B) Sequence of the goal locus for integration (high) aligned with the ssODN (backside) to introduce the Gaa^c1935C>A mutation (inexperienced field). PAM motifs are indicated in gold (gRNA-2) or blue (gRNA-3). Put in synonymous variants at PAM websites (Gaa^c.1920C>T, Gaa^c.1932G>A) and the specified KI mutation are highlighted in crimson. Put in gRNA seed area variants (Gaa^c.1923G>C, Gaa^c.1929G>A) are highlighted in inexperienced. (C) Sequencing chromatograms of management (Gaa^wt), founder #1 (Gaa^{c.1935 Founder #1}), and founder #2 (Gaa^{c.1935 Founder #2}). Black arrows point out synonymous variant edits at PAM websites (Gaa^c.1920, Gaa^c.1932) or gRNA seed areas (Gaa^c.1923, Gaa^c.1929). Crimson arrows point out the specified KI mutation (Gaa^c.1935C>A). Grey shaded area signifies amino acids at place 645 for every mouse. (D) WGS evaluation (> 50 × learn depth) of the Gaa^c.1935 locus in G₀ founder #1 and G₀ founder #2. WGS evaluation demonstrates extremely environment friendly on-target genome-editing in these founder mice. Knowledge are offered as stacked bar graphs indicating the proportion of WGS reads for every occasion class. Grey: nonspecific Gaa mutations; black: no Gaa mutation; crimson: supposed Gaa^c.1935C>A mutation and related synonymous variants; and blue: Gaa insertion/deletions. (E) Pedigree diagram of mating scheme to segregate the supposed Gaa^em1935C>A KI allele from mosaic CRISPR-generated founder mice for era of homozygous Gaa^em1935C>A KI mice. Males are represented as squares and females are represented as circles.

Founder #1 and founder #2 have been mated with WT animals, and their offspring G₁ HETs (male from founder #1 and feminine from founder #2) have been additional crossed to acquire the primary homozygous c.1935C>A KI mice within the G₂ era (Fig. 2E). Subsequently, mice harboring the c.1935C>A Gaa variant have been backcrossed 10 generations to the C57BL/6NJ background earlier than KI mice have been characterised. Because the era of our KI mice concerned CRISPR endonuclease-mediated mutation introduction, we adopted the Worldwide Committee on Standardized Genetic Nomenclature for Mice¹⁷ and named the KI transgenic mice as Gaa^em1935C>A.

Gaa
^em1935C>A KI mice have extreme GAA enzymatic deficiency and glycogen storage in cardiac, skeletal muscle, and mind tissue

The missense Gaa^c.1935C>A mutation in exon 14 of the Gaa gene results in an amino acid substitution; subsequently, we didn’t count on any nonsense-mediated decay in Gaa^c.1935C>A mRNA transcripts. The comparative ΔC_t between mouse Gaa and housekeeping gene Gapdh acquired by RT-PCR amongst WT, HET, and KI teams are virtually an identical, indicating the Gaa^c.1935C>A mutation doesn’t have an effect on Gaa mRNA ranges (Fig. 3A).

Molecular and biochemical characterization of Gaa^em1935C>A KI mice. (A) Gaa mRNA expression in tail or liver biopsy samples from 3-month-old WT (n = 4; black bar), HET (n = 8; striped bar), and KI (Gaa^em1935C>A; n = 6; white bar) mice Gaa expression ranges have been measured by TaqMan probe-based quantitative real-time PCR utilizing the ΔC_t methodology for comparability of the goal gene (Gaa) to the reference gene (Gapdh). The typical C_t worth from WT samples have been additional utilized to normalize with different teams. No vital distinction in Gaa mRNA transcript expression was detected amongst WT, HET, and KI samples. (B) GAA enzyme exercise in coronary heart, diaphragm, and gastrocnemius muscle tissues and mind homogenate from WT (n = 5; black bars), HET (n = 5; striped bars), KI (Gaa^em1935C>A; n = 4; white bars), and KO (Gaa^tm1Rabn; n = 3; gray bars) mice was measured utilizing a fluorometric 4-MU α-d-glucopyranoside assay and normalized to the quantity of pattern protein. (C) Glycogen stage was measured in the identical tissues used for evaluation in (B) utilizing a colorimetric assay. KO mice displayed considerably elevated glycogen ranges relative to WT and HET mice in all tissues assayed. Nevertheless, KI mice confirmed a big elevation of glycogen ranges in muscle tissues, however no vital elevation in mind. The quantity of glycogen was normalized to the quantity of pattern protein. Knowledge have been generated from a minimum of three unbiased experiments and proven as imply ± SD. All comparisons have been analyzed utilizing one-way ANOVA with the Tukey post-hoc take a look at. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns: not vital.

GAA enzymatic exercise was measured with synthetic fluorometric 4-MU substrate as described beforehand¹⁵. The outcomes have been according to the opposite findings from this research, exhibiting that the HET group had near 50% of the extent of enzymatic exercise noticed within the WT group in every muscle tissue and mind tissue pattern examined, indicating that the one WT allele produced useful enzyme, however not the c.1935C>A allele. For comparative functions, we acquired Gaa homozygous knock-out (KO) (B6;129-Gaa^tm1Rabn/J; exon 6 knock-out)¹⁸ mouse tissues from Jackson Laboratory (Bar Harbor, ME). In comparison with tissue from WT or HET animals, tissue from KI (Gaa^em1935C>A) and KO (Gaa^tm1Rabn/J) animals had considerably decreased GAA enzymatic exercise (about 1% of WT ranges) (Fig. 3B).

In comparison with the unaffected WT or HET teams, KI and KO mice had abnormally elevated lysosomal glycogen storage in coronary heart, diaphragm, and gastrocnemius muscle tissue. Curiously, elevated glycogen storage in whole-brain homogenates was noticed in KO mice, however not in KI mice, which had a slight, however not statistically vital, enhance in glycogen load (Fig. 3C).

Gaa
^em1935C>A KI mice present elevated muscle glycogen content material and elevated LAMP1 marker in mind areas

PAS staining is routinely used to reveal irregular carbohydrate accumulation in muscle tissue¹⁹. PAS staining was carried out in numerous muscle tissues (coronary heart, diaphragm, and gastrocnemius) from 3-month-old KI mice. Scattered crimson to magenta PAS staining particles representing the buildup of glycogen have been noticed in all three muscle tissue sorts within the KI mice, however not in WT animals (Fig. 4A). PAS staining with diastase (PAS-D), an enzyme that digests solely glycogen, was additionally utilized to consecutive slides to substantiate that the particles consisted of glycogen. A lower in crimson/magenta sign confirms that extreme accumulation merchandise in tissues comprised solely glycogen (Supplementary Fig. 1).

Tissue pathology in Gaa^em1935C>A KI mice exhibiting glycogen storage in muscle tissues and lysosomal abnormality in brains. (A) Consultant bright-field photos of coronary heart, diaphragm, and gastrocnemius sections from 3-month-old WT and KI mice, stained with hematoxylin/PAS. Areas of irregular glycogen accumulation (arrowheads) in cardiac and skeletal muscle tissues have been noticed in KI mice in comparison with WT mice (high). (B) Immunohistostaining with mouse anti-LAMP1 antibody exhibiting elevated cell physique staining (arrowheads) in frontal and hippocampal neurons and Purkinje cells of KI mice from all three consultant mind areas (frontal cortex, hippocampus and cerebellum). Scale bar represents 100 µm.

The lysosomal related membrane protein-1 (LAMP1) is usually used as a biomarker for lysosomal storage. LAMP1 staining within the mind sections from 3-month-old WT and KI mice have been examined in three consultant areas of the mind (frontal cortex, hippocampus, and cerebellum), demonstrating markedly elevated LAMP1 immunoreactivity in KI neuronal cell our bodies in comparison with WT controls (Fig. 4B).

In abstract, histopathology confirmed that the KI mice show early pathological glycogen accumulation in muscle tissues, which is analogous to muscle pathology in IOPD sufferers. As well as, the KI mice show a extra pronounced lysosomal burden within the mind areas as early as 3-months of age in comparison with WT animals.

Gaa
^em1935C>A KI mice have impaired skeletal muscle autophagy

Extreme autophagic buildup is well-documented in PD sufferers and in PD mice^20,21 and could also be a possible mechanism of PD pathogenesis. Microtubule-associated protein mild chain 3 (LC3B) is a protein element of autophagosomes, that are rapidly degraded underneath regular physiological circumstances and are hardly detectable. Cleavage of LC3B on the carboxy terminus instantly following synthesis yields the cytosolic, non-autophagosome certain LC3B-I kind. LC3B-I is transformed to autophagosome-bound LC3B-II through conjugation to phosphatidylethanolamine when autophagic processes are activated. Following autophagosome-lysosome fusion, LC3B-II is then hydrolyzed again to LC3B-I through ATG5²².

To look at autophagic standing of the Gaa^em1935C>A KI mice, western blotting for LC3B was carried out utilizing tissue homogenate (Fig. 5A and Supplementary Fig. 2). Each KI and KO fashions reveal elevated synthesis of LC3B-I in gastrocnemius, proof of upregulated autophagy (Fig. 5B); additional, autophagosomal LC3B-II is elevated in KI coronary heart, diaphragm, and gastrocnemius however not in mind (Fig. 5C). The ratio of LC3B-II:LC3B-I is elevated (Fig. 5D), demonstrating impaired autophagosome-lysosome fusion, in skeletal muscle tissues (diaphragm and gastrocnemius) however not cardiac muscle of the KI mannequin. That is an statement just like what has been noticed in each Gaa^em1826dupA KI and KO mouse fashions^15,21.

Autophagy impairment within the Gaa^c.1935C>A KI mouse mannequin. (A) Consultant western blot photos of autophagy-associated proteins (LC3B-I and LC3B-II) from tissue homogenate from coronary heart, diaphragm, gastrocnemius, and mind of WT (n = 3; black bars), HET (n = 3; striped bars), KI (n = 4; white bars), and Gaa^tm1Rabn (KO, n = 3; gray bars) mice. Outstanding LC3B-II bands may be seen in KI and KO tissues. (B) LC3B-I and (C) LC3B-II protein ranges normalized to the quantity of whole protein. Cytosolic LC3B-I is markedly elevated in KI and KO gastrocnemius muscle; autophagosomal LC3B-II is markedly elevated in KI and KO gastrocnemius, and reasonably elevated in KI coronary heart and diaphragm. (D) LC3B-II/LC3B-I ratio normalized to WT. Impaired autolysosomal formation (elevated LC3B-II/LC3B-I ratio) is noticed in KI skeletal muscle tissues. The ratio of LC3B-II and LC3B-I protein depth was quantified by densitometric evaluation of the western blots, and the ratio was additional normalized with WT in every tissue assayed. Knowledge have been generated from a minimum of three unbiased western blots and values are proven as imply ± SD. All comparisons have been analyzed utilizing one-way ANOVA with the Tukey post-hoc take a look at. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Gaa
^em1935C>A KI mice show left ventricular cardiac hypertrophy at 3 months of age

Neonatal-onset hypertrophic cardiomyopathy is a standard medical presentation in sufferers with IOPD. To discover the anatomical options and physiological operate of hearts within the KI mice, echocardiography was carried out on 3-month-old mice. M-mode photos obtained by echocardiography have been used to measure a number of parameters together with wall thickness, inner diameter, and coronary heart fee. Many extra useful parameters may be derived from these measurements to find out temporal left ventricular (LV) wall movement as an index for LV contractile patterns and chamber dimension (Fig. 6A).

Three-month-old Gaa^em1935C>A KI mice show anatomical options of left ventricular cardiac hypertrophy. (A) Consultant M-mode echocardiographic photos exhibiting cardiac dimensions: IVSd: yellow arrows; LVPWd: crimson arrows, and LVIDd / LVIDs: white arrows. (B) KI IVSd and LVPWd are considerably elevated versus WT and HET animals, indicative of concentric hypertrophic cardiomyopathy. (C) LVIDd and LVIDs don’t considerably differ between WT, HET, and KI animals. (D) KI fractional shortening is considerably elevated versus WT indicative of hyperdynamic contractility; KI LVMI is considerably elevated versus WT indicative once more of hypertrophic cardiomyopathy. All measurements from WT (n = 12; black bars), HET (n = 10; striped bars), and KI (Gaa^.em1935C>A; n = 10; white bars) mice have been obtained from 3-month-old mice. Knowledge are proven as imply ± SD. Coronary heart fee (HR) was maintained better than 500 bpm all through measurements. All comparisons have been analyzed utilizing one-way ANOVA with the Tukey post-hoc take a look at. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns: not vital.

Will increase in interventricular septal diameter (IVSd), LV posterior wall diameter (LVPWd), and LV mass index (LVMI) have been noticed in KI mice, in comparison with WT and/or HET mice (Fig. 6B), indicating pronounced hypertrophic cardiomyopathy. Measurements of myocardial contraction confirmed a slight lower in LV systolic inner diameter (LVIDs) within the KI mouse, however no vital distinction in LV diastolic inner diameter (LVIDd) was noticed amongst WT, HET, and KI mice (Fig. 6C). Elevated fractional shortening indicative of cardiac contractile dysfunction was noticed in KI mice (Fig. 6D). Echocardiographic knowledge subsequently signifies early hypertrophic cardiomyopathy phenotypes in 3-month-old Gaa^em1935C>A KI mice. The information offered in Fig. 5 present no gender variations in these parameters (Supplementary Fig. 3).

Diminished forelimb grip power in Gaa
^em1935C>A KI mice

The forelimb grip power take a look at is usually used to guage neuromuscular dysfunction in mice by measuring the deterioration of skeletal muscle. Peak stress drive was recorded because the mice misplaced their grip on the drive transducer bar and normalized to body weight for evaluation by gender group.

First, mouse physique weight is thought to vary between genders at 3 months of age²³. The imply ± SD physique weights of female and male mice in our research cohort have been 28.73 ± 3.13 g and 21.76 ± 2.36 g, respectively. In every gender cohort, there was no vital distinction in physique weight throughout WT, HET, and KI mice (Fig. 7). As well as, at 3 months of age, the male Gaa^em1935C>A KI mouse confirmed a big discount (~ 19%) in normalized peak stress drive in comparison with WT mice, indicating decreased forelimb muscle power in KI mice (Fig. 7). This discount was noticed solely in male KI mice, however not in feminine KI mice.

Diminished forelimb grip power in male Gaa^em1935C>A transgenic mice. Forelimb peak stress drive and physique mass measurements in 3-month-old male WT (n = 12; black bars), HET (n = 12; striped bars), and KI (n = 14; white bars) mice (high panel) and feminine WT (n = 12; black bars), HET (n = 12; striped bars), and KI (n = 11; white bars) mice (backside panel). No vital distinction in physique weight in every gender cohort amongst WT, HET and ki teams. Male KI mice reveal decreased normalized peak stress drive according to skeletal muscle weak point. Forelimb peak stress drive was measured utilizing a grip power meter and brought as the common of 9 trials over 3 days. Knowledge are proven as imply ± SD. All comparisons have been analyzed utilizing one-way ANOVA with the Tukey post-hoc take a look at. *p < 0.05, **p < 0.01, ***p < 0.001. ns: not vital.