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快速生成和筛选不具有脱靶效应的 Cas9 工程化 TRP53 R172P 小鼠。

Rapid generation and selection of Cas9-engineering TRP53 R172P mice that do not have off-target effects.

机构信息

The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen, 510275, Guangdong, China.

Tsinghua-Peking Center for Life Sciences, Beijing, 100084, China.

出版信息

BMC Biotechnol. 2019 Nov 8;19(1):74. doi: 10.1186/s12896-019-0573-z.

DOI:10.1186/s12896-019-0573-z
PMID:31703569
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6839086/
Abstract

BACKGROUND

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.

RESULTS

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.

CONCLUSIONS

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.

摘要

背景

基因突变导致严重的人类疾病，因此需要合适的动物模型来研究相关的调控机制。CRISPR/Cas9 系统是一种强大、高效且易于操作的遗传修饰工具。然而，利用 CRISPR/Cas9 在小鼠中引入点突变并排除脱靶效应仍然具有挑战性。TP53-R175 是人类癌症中最常突变的位点之一，它在包括癌症和糖尿病在内的人类疾病中发挥着关键作用。

结果

在这里，我们使用 CRISPR/Cas9 系统进行单次微注射，生成了 TRP53-R172P 突变小鼠（C57BL/6J，对应人类的 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 成分的浓度和注射的细胞部位。通过遗传和功能确认了 TRP53-R172P 的转换。TA 克隆和 Sanger 测序的组合有助于鉴定正确靶向的小鼠以及工程化小鼠中的脱靶效应，这为我们提供了一种快速高效选择无脱靶效应的靶小鼠的策略。

结论

单次注射这种优化的 CRISPR/Cas9 系统可用于在不产生脱靶效应的情况下在小鼠基因组中引入特定突变，从而模拟各种人类疾病。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/42f8/6839086/81e43a2ad7f7/12896_2019_573_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/42f8/6839086/a537cd5f9724/12896_2019_573_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/42f8/6839086/0d20c5f454ad/12896_2019_573_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/42f8/6839086/a62c01fef6cf/12896_2019_573_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/42f8/6839086/b8ed0e1aef4d/12896_2019_573_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/42f8/6839086/c64203452583/12896_2019_573_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/42f8/6839086/a836ba7eca0c/12896_2019_573_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/42f8/6839086/81e43a2ad7f7/12896_2019_573_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/42f8/6839086/a537cd5f9724/12896_2019_573_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/42f8/6839086/0d20c5f454ad/12896_2019_573_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/42f8/6839086/a62c01fef6cf/12896_2019_573_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/42f8/6839086/b8ed0e1aef4d/12896_2019_573_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/42f8/6839086/c64203452583/12896_2019_573_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/42f8/6839086/a836ba7eca0c/12896_2019_573_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/42f8/6839086/81e43a2ad7f7/12896_2019_573_Fig7_HTML.jpg

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