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CRISPR-Cas9 knockin mice for genome editing and cancer modeling. ​

Literature Information ​

PMID25263330
JournalCell
Impact Factor42.5
JCR QuartileQ1
Publication Year2014
Times Cited1070
KeywordsCRISPR-Cas9, genome editing, cancer modeling, mouse model, gene mutation
Literature TypeJournal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.
ISSN0092-8674
Pages440-55
Issue159(2)
AuthorsRandall J Platt, Sidi Chen, Yang Zhou, Michael J Yim, Lukasz Swiech, Hannah R Kempton, James E Dahlman, Oren Parnas, Thomas M Eisenhaure, Marko Jovanovic, Daniel B Graham, Siddharth Jhunjhunwala, Matthias Heidenreich, Ramnik J Xavier, Robert Langer, Daniel G Anderson, Nir Hacohen, Aviv Regev, Guoping Feng, Phillip A Sharp, Feng Zhang

TL;DR ​

This study introduces a Cre-dependent Cas9 knockin mouse to facilitate in vivo genome editing, demonstrating effective editing in various cell types using different delivery methods. The research highlights its application in modeling lung adenocarcinoma by generating mutations in key cancer-related genes, emphasizing the potential of Cas9 mice for diverse biological and disease research.

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CRISPR-Cas9 · genome editing · cancer modeling · mouse model · gene mutation

Abstract ​

CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras(G12D) mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.

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Primary Questions Addressed ​

  1. How can the use of Cre-dependent Cas9 knockin mice enhance our understanding of other cancer types beyond lung adenocarcinoma?
  2. What are the potential off-target effects of using CRISPR-Cas9 in vivo, particularly in the context of modeling complex diseases?
  3. In what ways can the delivery methods of guide RNA (AAV, lentivirus, particle-mediated) impact the efficiency of genome editing in different cell types?
  4. How might the findings from the CRISPR-Cas9 knockin mouse model influence future therapeutic strategies for treating cancers with mutations in KRAS, p53, and LKB1?
  5. What are the ethical considerations surrounding the use of genome editing technologies like CRISPR-Cas9 in animal models for cancer research?

Key Findings ​

Research Background and Objectives ​

CRISPR-Cas9 technology has revolutionized genome editing, allowing researchers to investigate genetic functions. The objective of this study was to develop a Cre-dependent Cas9 knockin mouse model to facilitate in vivo applications of Cas9 for studying genetic mutations associated with lung adenocarcinoma, particularly focusing on the genes KRAS, p53, and LKB1.

Main Methods/Materials/Experimental Design ​

The study employed a combination of in vivo and ex vivo genome editing techniques. The experimental design included the following key components:

  • Mouse Model Development: A Cre-dependent Cas9 knockin mouse was created to allow targeted editing of specific genes.
  • Delivery Systems: Various methods were utilized to deliver guide RNA, including:
    • Adeno-associated virus (AAV)
    • Lentivirus
    • Particle-mediated delivery
  • Target Cells: Editing was performed in different cell types, including neurons, immune cells, and endothelial cells.
  • Modeling Lung Adenocarcinoma: The researchers used the developed mouse model to introduce mutations in p53 and Lkb1 and to create Kras(G12D) mutations through homology-directed repair.

The following flowchart summarizes the technical route of the study:

Mermaid diagram

Key Results and Findings ​

  • Successful establishment of a Cre-dependent Cas9 knockin mouse model.
  • Demonstrated effective in vivo genome editing in multiple cell types using various delivery methods.
  • Induction of loss-of-function mutations in p53 and Lkb1, along with Kras(G12D) mutations, led to the development of macroscopic tumors resembling adenocarcinoma pathology.

Main Conclusions/Significance/Innovation ​

The study concludes that the Cre-dependent Cas9 knockin mouse model is a powerful tool for exploring the roles of specific genetic mutations in disease processes. The ability to model complex interactions among the top three mutated genes in lung adenocarcinoma (KRAS, p53, and LKB1) opens new avenues for research into cancer biology and therapeutic strategies.

Research Limitations and Future Directions ​

  • Limitations:

    • The study primarily focused on lung adenocarcinoma; further research is needed to assess the applicability of this model to other cancer types.
    • The long-term effects and potential off-target effects of CRISPR-Cas9 editing were not extensively evaluated.

  • Future Directions:

    • Investigate the utility of the Cas9 mouse model in studying other cancer-related genes and pathways.
    • Explore therapeutic interventions based on the findings of gene interactions and mutations.
    • Assess the long-term consequences of CRISPR-Cas9 editing in vivo to ensure safety and efficacy in potential clinical applications.

References ​

  1. High frequency of homologous recombination in mammalian cells between endogenous and introduced SV40 genomes. - M Jasin;J de Villiers;F Weber;W Schaffner - Cell (1985)
  2. Genome-scale CRISPR-Cas9 knockout screening in human cells. - Ophir Shalem;Neville E Sanjana;Ella Hartenian;Xi Shi;David A Scott;Tarjei Mikkelson;Dirk Heckl;Benjamin L Ebert;David E Root;John G Doench;Feng Zhang - Science (New York, N.Y.) (2014)
  3. DNA targeting specificity of RNA-guided Cas9 nucleases. - Patrick D Hsu;David A Scott;Joshua A Weinstein;F Ann Ran;Silvana Konermann;Vineeta Agarwala;Yinqing Li;Eli J Fine;Xuebing Wu;Ophir Shalem;Thomas J Cradick;Luciano A Marraffini;Gang Bao;Feng Zhang - Nature biotechnology (2013)
  4. Conditional control of gene expression in the mouse. - M Lewandoski - Nature reviews. Genetics (2001)
  5. Systematic discovery of TLR signaling components delineates viral-sensing circuits. - Nicolas Chevrier;Philipp Mertins;Maxim N Artyomov;Alex K Shalek;Matteo Iannacone;Mark F Ciaccio;Irit Gat-Viks;Elena Tonti;Marciela M DeGrace;Karl R Clauser;Manuel Garber;Thomas M Eisenhaure;Nir Yosef;Jacob Robinson;Amy Sutton;Mette S Andersen;David E Root;Ulrich von Andrian;Richard B Jones;Hongkun Park;Steven A Carr;Aviv Regev;Ido Amit;Nir Hacohen - Cell (2011)
  6. The AAV9 receptor and its modification to improve in vivo lung gene transfer in mice. - Christie L Bell;Luk H Vandenberghe;Peter Bell;Maria P Limberis;Guang-Ping Gao;Kim Van Vliet;Mavis Agbandje-McKenna;James M Wilson - The Journal of clinical investigation (2011)
  7. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. - Marina Bibikova;Mary Golic;Kent G Golic;Dana Carroll - Genetics (2002)
  8. Improved vectors and genome-wide libraries for CRISPR screening. - Neville E Sanjana;Ophir Shalem;Feng Zhang - Nature methods (2014)
  9. Transgenic expression of Cre recombinase from the tyrosine hydroxylase locus. - Jonas Lindeberg;Dmitry Usoskin;Henrik Bengtsson;Anna Gustafsson;Annika Kylberg;Stine Söderström;Ted Ebendal - Genesis (New York, N.Y. : 2000) (2004)
  10. Lung cancer. - Roy S Herbst;John V Heymach;Scott M Lippman - The New England journal of medicine (2008)

Literatures Citing This Work ​

  1. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. - Lukasz Swiech;Matthias Heidenreich;Abhishek Banerjee;Naomi Habib;Yinqing Li;John Trombetta;Mriganka Sur;Feng Zhang - Nature biotechnology (2015)
  2. Computational and molecular tools for scalable rAAV-mediated genome editing. - Ivaylo Stoimenov;Muhammad Akhtar Ali;Tatjana Pandzic;Tobias Sjöblom - Nucleic acids research (2015)
  3. Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi. - Manuel Kaulich;Yeon J Lee;Peter Lönn;Aaron D Springer;Bryan R Meade;Steven F Dowdy - Nucleic acids research (2015)
  4. Regenerative medicine: targeted genome editing in vivo. - Lixia Wang;Jun Wu;Weiwei Fang;Guang-Hui Liu;Juan Carlos Izpisua Belmonte - Cell research (2015)
  5. CRISPR-engineered mosaicism rapidly reveals that loss of Kcnj13 function in mice mimics human disease phenotypes. - Hua Zhong;Yiyun Chen;Yumei Li;Rui Chen;Graeme Mardon - Scientific reports (2015)
  6. Inducible in vivo genome editing with CRISPR-Cas9. - Lukas E Dow;Jonathan Fisher;Kevin P O'Rourke;Ashlesha Muley;Edward R Kastenhuber;Geulah Livshits;Darjus F Tschaharganeh;Nicholas D Socci;Scott W Lowe - Nature biotechnology (2015)
  7. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. - Mami Matano;Shoichi Date;Mariko Shimokawa;Ai Takano;Masayuki Fujii;Yuki Ohta;Toshiaki Watanabe;Takanori Kanai;Toshiro Sato - Nature medicine (2015)
  8. Genome editing strategies: potential tools for eradicating HIV-1/AIDS. - Kamel Khalili;Rafal Kaminski;Jennifer Gordon;Laura Cosentino;Wenhui Hu - Journal of neurovirology (2015)
  9. Dynamic interplay between bone and multiple myeloma: emerging roles of the osteoblast. - Michaela R Reagan;Lucy Liaw;Clifford J Rosen;Irene M Ghobrial - Bone (2015)
  10. The utility of rodent models of autism spectrum disorders. - Maria T Lázaro;Peyman Golshani - Current opinion in neurology (2015)

... (1060 more literatures)

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