Abstract
The clustered regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system functions like an adaptive immune system in a variety of microbes and has recently been engineered as a powerful tool for manipulating genomic sequences in a huge variety of cell types. In mammals, CRISPR/Cas9 has the potential to bring curative therapies to patients with genetic diseases, although it remained unknown whether suitable in vivo methods for its use are feasible. It is now appreciated that the efficient delivery of these genome-editing tools into most tissue types, including skin, remains a major challenge. Here, we describe a detailed protocol for performing in vivo gene editing of genomic sequences in mouse skin stem cells using Cas9/sgRNAs ribonucleoproteins in combination with electrotransfer technology. We here present all of the required methods needed for the protocol, including molecular cloning, in vitro sgRNA expression and sgRNA purification, Cas9 protein purification, and in vivo delivery of cas9 ribonucleoproteins. This protocol provides a novel in vivo gene editing strategy using ribonucleoproteins for skin stem cells and can potentially be used as curative treatment for genetic diseases in skin and other somatic tissues.
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References
Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327(5962):167
Bhaya D, Davison M, Barrangou R (2011) CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet 45:273–297
Cong L et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819
Jinek M et al (2013) RNA-programmed genome editing in human cells. Elife 2:e00471
Mali P et al (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823
Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213):1258096
Maddalo D et al (2014) In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature 516(7531):423–427
Yin H et al (2014) Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol 32(6):551–553
Swiech L et al (2015) In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol 33(1):102–106
Chiou SH et al (2015) Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing. Genes Dev 29(14):1576–1585
Bakondi B et al (2016) In vivo CRISPR/Cas9 gene editing corrects retinal dystrophy in the S334ter-3 rat model of autosomal dominant retinitis pigmentosa. Mol Ther 24(3):556–563
Long C et al (2016) Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 351(6271):400
Nelson CE et al (2016) In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351(6271):403
Maresch R et al (2016) Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice. Nat Commun 7:10770
Yarmush ML, Golberg A, Sersa G, Kotnik T, Miklavcic D (2014) Electroporation-based technologies for medicine: principles, applications, and challenges. Annu Rev Biomed Eng 16:295–320
Titomirov AV, Sukharev S, Kistanova E (1991) In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA. Biochim Biophys Acta 1088(1):131–134
Heller R et al (1996) In vivo gene electroinjection and expression in rat liver. FEBS Lett 389(3):225–228
Aihara H, Miyazaki J (1998) Gene transfer into muscle by electroporation in vivo. Nat Biotechnol 16(9):867–870. 1087-0156 (Print)
Heller LC, Heller R (2006) In vivo electroporation for gene therapy. Hum Gene Ther 17(9):890–897
Favard C, Dean D, Rols M-P (2007) Electrotransfer as a non viral method of gene delivery. Curr Gene Ther 7(1):67–77
Cemazar M, Sersa G (2007) Electrotransfer of therapeutic molecules into tissues. Curr Opin Mol Ther 9(6):554–562
Suzuki K et al (2016) In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540(7631):144–149
Wu W (2016) Efficient in vivo gene editing using ribonucleoproteins in skin stem cells of recessive dystrophic epidermolysis bullosa mouse model. Proc Natl Acad Sci U S A 114(7):1660–1665
Ran FA et al (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281–2308
Cong L, Zhang F (2015) Genome engineering using CRISPR-Cas9 system. Methods Mol Biol 1239:197–217
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Wu, W., Chen, T. (2018). Ribonucleoproteins Mediated Efficient In Vivo Gene Editing in Skin Stem Cells. In: Turksen, K. (eds) Skin Stem Cells. Methods in Molecular Biology, vol 1879. Humana Press, New York, NY. https://doi.org/10.1007/7651_2018_115
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DOI: https://doi.org/10.1007/7651_2018_115
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8869-3
Online ISBN: 978-1-4939-8870-9
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