One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering

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.

[1]  N. Copeland,et al.  Harnessing transposons for cancer gene discovery , 2010, Nature Reviews Cancer.

[2]  Z. Deng,et al.  The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes , 2011, Nature.

[3]  Jaap Kool,et al.  High throughput insertional mutagenesis screens in mice to identify oncogenic networks , 2009, Nature Reviews Cancer.

[4]  O. Abdel-Wahab,et al.  Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation. , 2011, Cancer cell.

[5]  Shondra M Pruett-Miller,et al.  High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases , 2011, Nature Methods.

[6]  Wolfgang Wurst,et al.  Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides , 2013, Proceedings of the National Academy of Sciences.

[7]  Jeffrey C. Miller,et al.  A rapid and general assay for monitoring endogenous gene modification. , 2010, Methods in molecular biology.

[8]  W. Wurst,et al.  Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases , 2010, Proceedings of the National Academy of Sciences.

[9]  Mario R. Capecchi,et al.  Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century , 2005, Nature Reviews Genetics.

[10]  Frank Lyko,et al.  Combined deficiency of Tet1 and Tet2 causes epigenetic abnormalities but is compatible with postnatal development. , 2013, Developmental cell.

[11]  A. Bogdanove,et al.  TAL Effectors: Customizable Proteins for DNA Targeting , 2011, Science.

[12]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[13]  Seung Woo Cho,et al.  Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease , 2013, Nature Biotechnology.

[14]  Xiaojun Zhu,et al.  Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos , 2013, Cell Research.

[15]  J. Doudna,et al.  RNA-guided genetic silencing systems in bacteria and archaea , 2012, Nature.

[16]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[17]  Jun Zhang,et al.  Generation of gene-modified mice via Cas9/RNA-mediated gene targeting , 2013, Cell Research.

[18]  Susan Lindquist,et al.  Generation of Isogenic Pluripotent Stem Cells Differing Exclusively at Two Early Onset Parkinson Point Mutations , 2011, Cell.

[19]  D. Carroll,et al.  Gene targeting in Drosophila and Caenorhabditis elegans with zinc-finger nucleases. , 2008, Methods in molecular biology.

[20]  L. Liaw,et al.  Targeted Genome Modification in Mice Using Zinc-Finger Nucleases , 2010, Genetics.

[21]  Elo Leung,et al.  Knockout rats generated by embryo microinjection of TALENs , 2011, Nature Biotechnology.

[22]  Jeffry D. Sander,et al.  Efficient In Vivo Genome Editing Using RNA-Guided Nucleases , 2013, Nature Biotechnology.

[23]  L. Symington,et al.  Double-strand break end resection and repair pathway choice. , 2011, Annual review of genetics.

[24]  R. Barrangou,et al.  CRISPR/Cas, the Immune System of Bacteria and Archaea , 2010, Science.

[25]  Han-Woong Lee,et al.  Knockout mice created by TALEN-mediated gene targeting , 2013, Nature Biotechnology.

[26]  Feng-Chun Yang,et al.  Deletion of Tet2 in mice leads to dysregulated hematopoietic stem cells and subsequent development of myeloid malignancies. , 2011, Blood.

[27]  Jaap Kool,et al.  High-throughput insertional mutagenesis screens in mice to identify oncogenic networks , 2009, Nature Reviews Cancer.

[28]  Ignacio Anegon,et al.  Knockout Rats via Embryo Microinjection of Zinc-Finger Nucleases , 2009, Science.

[29]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[30]  D. Page,et al.  Tet1 is dispensable for maintaining pluripotency and its loss is compatible with embryonic and postnatal development. , 2011, Cell stem cell.

[31]  Feng Zhang,et al.  CRISPR-assisted editing of bacterial genomes , 2013, Nature Biotechnology.

[32]  X. Cui,et al.  Targeted integration in rat and mouse embryos with zinc-finger nucleases , 2011, Nature Biotechnology.

[33]  E. Rebar,et al.  Genome editing with engineered zinc finger nucleases , 2010, Nature Reviews Genetics.

[34]  R. Barrangou,et al.  Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria , 2012, Proceedings of the National Academy of Sciences.

[35]  M. McVey,et al.  MMEJ repair of double-strand breaks (director's cut): deleted sequences and alternative endings. , 2008, Trends in genetics : TIG.