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.

中文翻译：

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快速产生和选择没有脱靶效应的Cas9工程TRP53 R172P小鼠

基因突变会导致严重的人类疾病，因此需要合适的动物模型来研究所涉及的调节机制。CRISPR / Cas9系统是一种功能强大，高效且易于操作的基因修饰工具。然而，利用CRISPR / Cas9引入点突变以及排除小鼠脱靶效应仍然具有挑战性。TP53-R175是人类癌症中最常见的突变位点之一，在人类疾病（包括癌症和糖尿病）中起着至关重要的作用。在这里，我们使用CRISPR / Cas9系统的单次显微注射生成了TRP53-R172P突变小鼠（C57BL / 6 J，对应于人类中的TP53-R175P）。最佳参数包括gRNA选择，供体指定（gRNA区域内的沉默突变），CRISPR组分的浓度和注射的细胞部位。TRP53-R172P转换在基因和功能上得到确认。TA克隆和Sanger测序的组合有助于识别正确靶向的小鼠以及工程小鼠的脱靶效应，这为我们提供了一种策略，可以快速，有效地选择无脱靶效应的靶上小鼠。这种优化的CRISPR / Cas9系统的单次注射可用于在小鼠基因组中引入特定的突变，而不会产生脱靶效应，从而可以模拟各种人类疾病。