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The c.1935C&gt;A (p.Asp645Glu) variant, the most frequent <jats:italic>GAA<\/jats:italic> pathogenic mutation in people of Southern Han Chinese ancestry, causes infantile-onset Pompe disease (IOPD), presenting neonatally with severe hypertrophic cardiomyopathy, profound muscle hypotonia, respiratory failure, and infantile mortality. We applied CRISPR-Cas9 homology-directed repair (HDR) using a novel dual sgRNA approach flanking the target site to generate a <jats:italic>Gaa<\/jats:italic><jats:sup><jats:italic>em1935C<\/jats:italic>&gt;<jats:italic>A<\/jats:italic><\/jats:sup> knock-in mouse model and a myoblast cell line carrying the <jats:italic>Gaa<\/jats:italic> c.1935C&gt;A mutation. Herein we describe the molecular, biochemical, histological, physiological, and behavioral characterization of 3-month-old homozygous <jats:italic>Gaa<\/jats:italic><jats:sup><jats:italic>em1935C<\/jats:italic>&gt;<jats:italic>A<\/jats:italic><\/jats:sup> mice. Homozygous <jats:italic>Gaa<\/jats:italic><jats:sup><jats:italic>em1935C<\/jats:italic>&gt;<jats:italic>A<\/jats:italic><\/jats:sup> knock-in mice exhibited normal <jats:italic>Gaa<\/jats:italic> mRNA expression levels relative to wild-type mice, had near-abolished GAA enzymatic activity, markedly increased tissue glycogen storage, and concomitantly impaired autophagy. Three-month-old mice demonstrated skeletal muscle weakness and hypertrophic cardiomyopathy but no premature mortality. The <jats:italic>Gaa<\/jats:italic><jats:sup><jats:italic>em1935C<\/jats:italic>&gt;<jats:italic>A<\/jats:italic><\/jats:sup> knock-in mouse model recapitulates multiple salient aspects of human IOPD caused by the <jats:italic>GAA<\/jats:italic> c.1935C&gt;A pathogenic variant. It is an ideal model to assess innovative therapies to treat IOPD, including personalized therapeutic strategies that correct pathogenic variants, restore GAA activity and produce functional phenotypes.<\/jats:p>","DOI":"10.1038\/s41598-022-25914-8","type":"journal-article","created":{"date-parts":[[2022,12,14]],"date-time":"2022-12-14T11:06:27Z","timestamp":1671015987000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["CRISPR-mediated generation and characterization of a Gaa homozygous c.1935C&gt;A (p.D645E) Pompe disease knock-in mouse model recapitulating human infantile onset-Pompe disease"],"prefix":"10.1038","volume":"12","author":[{"given":"Shih-hsin","family":"Kan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeffrey Y.","family":"Huang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jerry","family":"Harb","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Allisandra","family":"Rha","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nancy D.","family":"Dalton","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chloe","family":"Christensen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yunghang","family":"Chan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeremy","family":"Davis-Turak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan","family":"Neumann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raymond Y.","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,12,14]]},"reference":[{"issue":"2","key":"25914_CR1","doi-asserted-by":"publisher","first-page":"241","DOI":"10.1016\/j.resp.2013.06.007","volume":"189","author":"DD Fuller","year":"2013","unstructured":"Fuller, D. 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