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

Literature Information ​

PMID23643243
JournalCell
Impact Factor42.5
JCR QuartileQ1
Publication Year2013
Times Cited1900
KeywordsCRISPR/Cas system, gene editing, mouse mutations
Literature TypeJournal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't
ISSN0092-8674
Pages910-8
Issue153(4)
AuthorsHaoyi Wang, Hui Yang, Chikdu S Shivalila, Meelad M Dawlaty, Albert W Cheng, Feng Zhang, Rudolf Jaenisch

TL;DR ​

This study demonstrates the efficiency of the CRISPR/Cas system in generating mice with simultaneous mutations in multiple genes, specifically achieving high disruption rates in five genes using coinjection strategies. This innovative approach not only streamlines the creation of genetically modified animals but also enhances the ability to investigate functionally redundant genes and their interactions, significantly advancing genetic research.

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CRISPR/Cas system · gene editing · mouse mutations

Abstract ​

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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Primary Questions Addressed ​

  1. What are the potential implications of using CRISPR/Cas for studying gene interactions in disease models?
  2. How does the efficiency of CRISPR/Cas-mediated gene editing compare to traditional methods for generating multiple gene mutations?
  3. What challenges might arise in the application of CRISPR/Cas technology for multiplexed genome editing in other organisms?
  4. In what ways can the one-step generation of genetically modified mice contribute to understanding complex traits and behaviors?
  5. How can the precise point mutations generated by CRISPR/Cas impact the study of gene function and regulation?

Key Findings ​

Research Background and Objectives ​

Traditional methods for generating mice with multiple gene mutations involve labor-intensive processes such as sequential recombination in embryonic stem cells and extensive intercrossing of single-mutant mice. This study aims to demonstrate the efficacy of the CRISPR/Cas system as a novel and efficient approach for multiplexed genome editing, enabling the simultaneous disruption of multiple genes in a single step.

Main Methods/Materials/Experimental Design ​

The research employs the CRISPR/Cas gene-editing technology, focusing on the following key steps:

  1. CRISPR/Cas System: Utilizes Cas9 protein and single-guide RNAs (sgRNAs) for targeted gene disruption.
  2. Target Genes: The study targets five genes: Tet1, Tet2, Tet3, Sry, and Uty.
  3. Embryonic Stem Cells: Mouse embryonic stem (ES) cells are used for initial gene editing.
  4. Zygote Injection: Coinjection of Cas9 mRNA and sgRNAs into zygotes is performed to achieve biallelic mutations.

The technical workflow can be represented as follows:

Mermaid diagram

Key Results and Findings ​

  • High Efficiency: The CRISPR/Cas system allowed for the simultaneous disruption of five genes with high efficiency in ES cells.
  • Biallelic Mutations: Coinjection of Cas9 mRNA and sgRNAs targeting Tet1 and Tet2 resulted in 80% efficiency for generating biallelic mutations.
  • Precise Mutations: The method also enabled the introduction of precise point mutations in two target genes when combined with mutant oligos.

Main Conclusions/Significance/Innovation ​

The study demonstrates that the CRISPR/Cas system can facilitate the one-step generation of mice with mutations in multiple genes, which significantly streamlines the process of genetic manipulation in research. This innovative approach is expected to enhance the study of functionally redundant genes and epistatic interactions, ultimately advancing our understanding of complex genetic networks.

Research Limitations and Future Directions ​

  • Limitations: The study primarily focuses on a limited number of genes, and the long-term effects of these mutations in vivo remain to be fully characterized. Additionally, the efficiency may vary depending on the specific gene targets and their genomic contexts.
  • Future Directions: Further research should explore the application of this technology to a broader range of genes and biological systems. Investigating the functional consequences of these mutations in various models will be crucial for understanding their roles in development and disease. Additionally, optimizing the CRISPR/Cas system for improved precision and reduced off-target effects will be important for future applications.

References ​

  1. Knockout mice created by TALEN-mediated gene targeting. - Young Hoon Sung;In-Jeoung Baek;Duk Hyoung Kim;Jisun Jeon;Jaehoon Lee;Kyunghee Lee;Daewon Jeong;Jin-Soo Kim;Han-Woong Lee - Nature biotechnology (2013)
  2. Efficient genome editing in zebrafish using a CRISPR-Cas system. - Woong Y Hwang;Yanfang Fu;Deepak Reyon;Morgan L Maeder;Shengdar Q Tsai;Jeffry D Sander;Randall T Peterson;J-R Joanna Yeh;J Keith Joung - Nature biotechnology (2013)
  3. The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. - Tian-Peng Gu;Fan Guo;Hui Yang;Hai-Ping Wu;Gui-Fang Xu;Wei Liu;Zhi-Guo Xie;Linyu Shi;Xinyi He;Seung-gi Jin;Khursheed Iqbal;Yujiang Geno Shi;Zixin Deng;Piroska E Szabó;Gerd P Pfeifer;Jinsong Li;Guo-Liang Xu - Nature (2011)
  4. Double-strand break end resection and repair pathway choice. - Lorraine S Symington;Jean Gautier - Annual review of genetics (2011)
  5. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. - Wenyan Jiang;David Bikard;David Cox;Feng Zhang;Luciano A Marraffini - Nature biotechnology (2013)
  6. Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides. - Benedikt Wefers;Melanie Meyer;Oskar Ortiz;Martin Hrabé de Angelis;Jens Hansen;Wolfgang Wurst;Ralf Kühn - Proceedings of the National Academy of Sciences of the United States of America (2013)
  7. Deletion of Tet2 in mice leads to dysregulated hematopoietic stem cells and subsequent development of myeloid malignancies. - Zhe Li;Xiaoqiang Cai;Chen-Leng Cai;Jiapeng Wang;Wenyong Zhang;Bruce E Petersen;Feng-Chun Yang;Mingjiang Xu - Blood (2011)
  8. Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. - Frank Soldner;Josée Laganière;Albert W Cheng;Dirk Hockemeyer;Qing Gao;Raaji Alagappan;Vikram Khurana;Lawrence I Golbe;Richard H Myers;Susan Lindquist;Lei Zhang;Dmitry Guschin;Lauren K Fong;B Joseph Vu;Xiangdong Meng;Fyodor D Urnov;Edward J Rebar;Philip D Gregory;H Steve Zhang;Rudolf Jaenisch - Cell (2011)
  9. Combined deficiency of Tet1 and Tet2 causes epigenetic abnormalities but is compatible with postnatal development. - Meelad M Dawlaty;Achim Breiling;Thuc Le;Günter Raddatz;M Inmaculada Barrasa;Albert W Cheng;Qing Gao;Benjamin E Powell;Zhe Li;Mingjiang Xu;Kym F Faull;Frank Lyko;Rudolf Jaenisch - Developmental cell (2013)
  10. TAL effectors: customizable proteins for DNA targeting. - Adam J Bogdanove;Daniel F Voytas - Science (New York, N.Y.) (2011)

Literatures Citing This Work ​

  1. A coupled protein and probe engineering approach for selective inhibition and activity-based probe labeling of the caspases. - Junpeng Xiao;Petr Broz;Aaron W Puri;Edgar Deu;Montse Morell;Denise M Monack;Matthew Bogyo - Journal of the American Chemical Society (2013)
  2. Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. - Scott J Gratz;Alexander M Cummings;Jennifer N Nguyen;Danielle C Hamm;Laura K Donohue;Melissa M Harrison;Jill Wildonger;Kate M O'Connor-Giles - Genetics (2013)
  3. Progress and prospects in stem cell therapy. - Xiu-ling Xu;Fei Yi;Hui-ze Pan;Shun-lei Duan;Zhi-chao Ding;Guo-hong Yuan;Jing Qu;Hai-chen Zhang;Guang-hui Liu - Acta pharmacologica Sinica (2013)
  4. Novel GM animal technologies and their governance. - Ann Bruce;David Castle;Corrina Gibbs;Joyce Tait;C Bruce A Whitelaw - Transgenic research (2013)
  5. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. - Yanfang Fu;Jennifer A Foden;Cyd Khayter;Morgan L Maeder;Deepak Reyon;J Keith Joung;Jeffry D Sander - Nature biotechnology (2013)
  6. Rad51, friend or foe? - Sue Mei Tan-Wong;Nick J Proudfoot - eLife (2013)
  7. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. - Ari E Friedland;Yonatan B Tzur;Kevin M Esvelt;Monica P Colaiácovo;George M Church;John A Calarco - Nature methods (2013)
  8. 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)
  9. 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)
  10. Overcoming barriers and thresholds - signaling of oligomeric Aβ through the prion protein to Fyn. - Hansen Wang;Carl He Ren;C Geeth Gunawardana;Gerold Schmitt-Ulms - Molecular neurodegeneration (2013)

... (1890 more literatures)

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