Abstract
BackgroundGenetic 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.ResultsHere, 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.ConclusionsA 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.
Highlights
Genetic mutations cause severe human diseases, and suitable animal models to study the regulatory mechanisms involved are required
(See figure on previous page.) Fig. 1 Introduction of the R172P substitution in TRP53 locus via a single injection of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system. a Procedure of construction of KI mice: Step 1) Design gRNA and donor for mouse engineering; Step 2) In vitro transcription to generate mRNA of Cas9 and gRNA; Step 3) Prepare zygotes for microinjection and inject Cas9 mRNA, gRNA and donor into zygotes as well as transplant obtained zygotes into foster mother mice. b A schematic illustration shows the designation of gRNA and donor in TRP53 R172P KI mouse engineering
The mouse model of this TP53 mutant escapes the early onset of spontaneous tumorigenesis [10] but develops diabetes [11] as well as colon adenocarcinomas [12] upon the deficiency of nonhomologous endjoining (NHEJ). These findings suggest that the mouse model of the human TP53 R175P mutant is valuable to explore the influences of TP53’s ability of cell cycle arrest in human diseases, including cancer and diabetes
Summary
Genetic mutations cause severe human diseases, and suitable animal models to study the regulatory mechanisms involved are required. Suitable animal models are urgently needed to elucidate the regulatory mechanisms of genetic mutations in the development and progression of human diseases. The donor carried 6 silent mutations, which do not cause amino acid replacement, in the gRNA region and a G- > C mutation to generate the R172P substitution of the TRP53 tumour suppressor (bottom panel). PCR amplification of hSpCas cDNA from the pX260 vector was performed using the Phusion high-fidelity PCR kit, and the single band product with the correct length is shown in the agarose gel (left panel). PCR amplification of gRNA DNA with high-fidelity PCR kit resulted in a single band product with the correct length as shown in the agarose gel (left panel). The in vitro transcription product of gRNA was validated by agarose gel electrophoresis (right panel)
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