In vivo homology directed repair in heart, skeletal muscle, and muscle stem cells
A heart cell, muscle technology, applied in the field of in vivo homology-directed repair in heart, skeletal muscle and muscle stem cells, can solve the problems of untested multi-organ HDR feasibility and inefficiency
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[0113] To sensitively detect in vivo gene editing events via CRISPR / Cas9, a strongly enhanced green fluorescent protein (GFP) signal using ubiquitous expression was developed 8 A fluorescent protein-based reporter system for transgenic mouse strains ( Figure 1A ). Based on published BFP variants 9-11 The blue fluorescent protein (BFP) sequence was designed to carry a minimum of 2 base substitutions (C197G and T199C) compared to the GFP sequence. This simple modification allows easy differentiation of two fluorescent proteins by fluorescence-activated cell sorting (FACS) ( Figure 5A-Figure 5D ). The same 2 base substitutions also created a Btgl site for restriction fragment length polymorphism (RFLP) analysis. A single guide RNA (sgRNA) targeting a substitution site in GFP was designed to be compatible with the Cas9 protein (SaCas9) from S. + / - ; Efficient disruption of GFP signaling was tested in tail tip fibroblasts (TTF) of mdx mice ( Figure 5B , Figure 5C ). Thi...
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