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• W2988173622 abstract "Genetic mutations cause severe human diseases, and suitable animal models to study the regulatory mechanisms involved are required. The CRISPR/Cas9 system is a powerful, highly efficient and easily manipulated tool for genetic modifications. However, utilization of CRISPR/Cas9 to introduce point mutations and the exclusion of off-target effects in mice remain challenging. TP53-R175 is one of the most frequently mutated sites in human cancers, and it plays crucial roles in human diseases, including cancers and diabetes.Here, we generated TRP53-R172P mutant mice (C57BL/6 J, corresponding to TP53-R175P in humans) using a single microinjection of the CRISPR/Cas9 system. The optimal parameters comprised gRNA selection, donor designation (silent mutations within gRNA region), the concentration of CRISPR components and the cellular sites of injection. TRP53-R172P conversion was genetically and functionally confirmed. Combination of TA cloning and Sanger sequencing helped identify the correctly targeted mice as well as the off-target effects in the engineered mice, which provide us a strategy to select the on-target mice without off-target effects quickly and efficiently.A single injection of the this optimized CRISPR/Cas9 system can be applied to introduce particular mutations in the genome of mice without off-target effects to model various human diseases." @default.
• W2988173622 created "2019-11-22" @default.
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• W2988173622 date "2019-11-08" @default.
• W2988173622 modified "2023-10-06" @default.
• W2988173622 title "Rapid generation and selection of Cas9-engineering TRP53 R172P mice that do not have off-target effects" @default.
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• W2988173622 doi "https://doi.org/10.1186/s12896-019-0573-z" @default.
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