Background: Genetic mutations cause severe human diseases, and suitable animal models to study the regulatory\nmechanisms involved are required. The CRISPR/Cas9 system is a powerful, highly efficient and easily manipulated\ntool for genetic modifications. However, utilization of CRISPR/Cas9 to introduce point mutations and the exclusion\nof off-target effects in mice remain challenging. TP53-R175 is one of the most frequently mutated sites in human\ncancers, and it plays crucial roles in human diseases, including cancers and diabetes.\nResults: Here, we generated TRP53-R172P mutant mice (C57BL/6 J, corresponding to TP53-R175P in humans) using\na single microinjection of the CRISPR/Cas9 system. The optimal parameters comprised gRNA selection, donor\ndesignation (silent mutations within gRNA region), the concentration of CRISPR components and the cellular sites\nof injection. TRP53-R172P conversion was genetically and functionally confirmed. Combination of TA cloning and\nSanger sequencing helped identify the correctly targeted mice as well as the off-target effects in the engineered\nmice, which provide us a strategy to select the on-target mice without off-target effects quickly and efficiently.\nConclusions: A single injection of the this optimized CRISPR/Cas9 system can be applied to introduce particular\nmutations in the genome of mice without off-target effects to model various human diseases.
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