1. Novel reporter mouse models useful for evaluating in vivo gene editing and for optimization of methods of delivering genome editing tools
- Author
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Hiromi Miura, Akiko Mizutani, Aki Kurosaki, Masato Ohtsuka, Masahiro Sato, Guisheng Zhang, Dexi Liu, Yongjie Ma, Kenya Kamimura, Jurai Imafuku, and Channabasavaiah B. Gurumurthy
- Subjects
0301 basic medicine ,Genetically modified mouse ,in vivo gene editing ,Cas9 ,Genetic enhancement ,EGFP ,RM1-950 ,Computational biology ,transgenic mice ,Biology ,gene therapy ,Genome ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Genome editing ,CRISPR ,030220 oncology & carcinogenesis ,Drug Discovery ,genome editing efficiency ,Molecular Medicine ,Therapeutics. Pharmacology ,Guide RNA ,Ribonucleoprotein - Abstract
The clustered regularly interspersed palindromic repeats (CRISPR) system is a powerful genome-editing tool to modify genomes, virtually in any species. The CRISPR tool has now been utilized in many areas of medical research, including gene therapy. Although several proof-of-concept studies show the feasibility of in vivo gene therapy applications for correcting disease-causing mutations, and new and improved tools are constantly being developed, there are not many choices of suitable reporter models to evaluate genome editor tools and their delivery methods. Here, we developed and validated reporter mouse models containing a single copy of disrupted EGFP (ΔEGFP) via frameshift mutations. We tested several delivery methods for validation of the reporters, and we demonstrated their utility to assess both non-homologous end-joining (NHEJ) and via homology-directed repair (HDR) processes in embryos and in somatic tissues. With the use of the reporters, we also show that hydrodynamic delivery of ribonucleoprotein (RNP) with Streptococcus pyogenes (Sp)Cas9 protein mixed with synthetic guide RNA (gRNA) elicits better genome-editing efficiencies than the plasmid vector-based system in mouse liver. The reporters can also be used for assessing HDR efficiencies of the Acidaminococcus sp. (As)Cas12a nuclease. The results suggest that the ΔEGFP mouse models serve as valuable tools for evaluation of in vivo genome editing.
- Published
- 2021