One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.

文献信息

PMID	23643243
期刊	Cell
影响因子	42.5
JCR 分区	Q1
发表年份	2013
被引次数	1900
关键词	CRISPR/Cas系统, 基因编辑, 小鼠突变, 多基因, 胚胎干细胞
文献类型	Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't
ISSN	0092-8674
页码	910-8
期号	153(4)
作者	Haoyi Wang, Hui Yang, Chikdu S Shivalila, Meelad M Dawlaty, Albert W Cheng, Feng Zhang, Rudolf Jaenisch

一句话小结

本研究利用CRISPR/Cas系统实现了在小鼠胚胎干细胞中高效同时破坏五个基因，生成双等位基因突变的小鼠，突变效率高达80%。该方法不仅显著简化了多基因突变小鼠的生成过程，还将加速对功能冗余基因及其表观基因相互作用的研究。

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CRISPR/Cas系统 · 基因编辑 · 小鼠突变 · 多基因 · 胚胎干细胞

摘要

携带多个基因突变的小鼠传统上是通过在胚胎干细胞中进行顺序重组和/或耗时的单一突变小鼠的交配来生成的。CRISPR/Cas系统已被改编为一种高效的基因靶向技术，具有多重基因组编辑的潜力。我们展示了CRISPR/Cas介导的基因编辑能够以高效率同时破坏小鼠胚胎干细胞中的五个基因（Tet1、Tet2、Tet3、Sry、Uty——共8个等位基因）。将Cas9 mRNA与针对Tet1和Tet2的单导RNA（sgRNA）共同注射到合子中，生成了这两个基因具有双等位基因突变的小鼠，效率达80%。最后，我们展示了将Cas9 mRNA/sgRNA与突变寡核苷酸共同注射，可以同时在两个靶基因中生成精确的点突变。因此，CRISPR/Cas系统允许一步生成携带多个基因突变的动物，这一方法将大大加速对功能冗余基因及其表观基因相互作用的体内研究。

英文摘要

Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty--8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.

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主要研究问题

核心洞察

研究背景和目的

传统上，通过在胚胎干细胞中进行序列重组或耗时的单基因小鼠交配来生成携带多基因突变的小鼠。这种方法效率低下且耗时。CRISPR/Cas系统作为一种高效的基因靶向技术，具有多重基因组编辑的潜力。本研究旨在展示CRISPR/Cas介导的基因编辑技术在小鼠胚胎干细胞中同时破坏多个基因的能力，从而加速对功能冗余基因和表观遗传基因相互作用的体内研究。

主要方法/材料/实验设计

本研究采用CRISPR/Cas系统进行基因编辑，具体步骤如下：

设计sgRNA：针对目标基因Tet1、Tet2、Tet3、Sry和Uty设计单导向RNA（sgRNA）。
合成Cas9 mRNA：合成编码Cas9蛋白的mRNA。
注射：将Cas9 mRNA和sgRNA共同注射到小鼠受精卵中。
筛选突变小鼠：通过PCR和测序筛选出具有双等位基因突变的小鼠，达到80%的效率。
注射突变寡核苷酸：与sgRNA共同注射突变寡核苷酸，以实现两个目标基因的精确点突变。

关键结果和发现

使用CRISPR/Cas系统，研究成功地在小鼠胚胎干细胞中同时破坏了五个基因，突变效率高。
通过共同注射Cas9 mRNA和sgRNA，成功生成了具有双等位基因突变的小鼠，效率高达80%。
采用突变寡核苷酸与sgRNA共同注射的方式，实现了两个目标基因的精确点突变。

主要结论/意义/创新性

本研究表明，CRISPR/Cas系统可以在一个步骤中生成携带多个基因突变的小鼠，这种方法显著提高了多基因突变小鼠的生成效率。这一技术的应用将极大地推动对功能冗余基因及其相互作用的研究，提供了新的实验工具和策略，为生物医学研究开辟了新的方向。

研究局限性和未来方向

局限性：本研究主要集中在小鼠模型，尚需在其他物种中验证该技术的适用性和效率。
未来方向：建议进一步探索CRISPR/Cas系统在不同生物体中的应用，及其在基因治疗和疾病模型建立中的潜力。此外，优化sgRNA设计和提高突变效率仍然是未来研究的重点。

参考文献

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Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. - Andrew R Bassett;Charlotte Tibbit;Chris P Ponting;Ji-Long Liu - Cell reports (2013)
CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. - Luke A Gilbert;Matthew H Larson;Leonardo Morsut;Zairan Liu;Gloria A Brar;Sandra E Torres;Noam Stern-Ginossar;Onn Brandman;Evan H Whitehead;Jennifer A Doudna;Wendell A Lim;Jonathan S Weissman;Lei S Qi - Cell (2013)
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... (1890 更多篇文献)

One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. ​

文献信息 ​

一句话小结 ​

在麦伴科研 (maltsci.com) 搜索更多文献 ​

摘要 ​

英文摘要 ​

麦伴智能科研服务 ​

主要研究问题 ​

核心洞察 ​

研究背景和目的 ​

主要方法/材料/实验设计 ​

关键结果和发现 ​

主要结论/意义/创新性 ​

研究局限性和未来方向 ​

参考文献 ​

引用本文的文献 ​