Abstract
Huntington’s disease (HD) is a hereditary neurological disorder caused by expansion of the CAG repeat tract in the huntingtin gene (HTT). The mutant protein with a long polyglutamine tract is toxic to cells, especially neurons, leading to their progressive degeneration. Similar to many other monogenic diseases, HD is a good target for gene therapy approaches, including the use of programmable endonucleases. Here, we describe a protocol for HTT gene knock out using a modified Cas9 protein (nickase, Cas9n) and a pair of sgRNAs flanking the repeats. Recently, we showed that excision of the CAG repeat tract resulted in a frameshift mutation and premature translation termination. As a model, we used HD patient-derived fibroblasts electroporated with a pair of plasmid vectors expressing CRISPR-Cas9n tools. Efficient HTT inactivation independent of the CAG tract length was confirmed by Western blotting. A modified version of this protocol involving precise excision of the CAG repeats and insertion of a new DNA sequence by homology directed repair may also be used for the generation of new isogenic cellular models of HD.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Bates GP, Dorsey R, Gusella JF et al (2015) Huntington disease. Nat Rev Dis Primers 1:15005. https://doi.org/10.1038/nrdp.2015.5
Wild EJ, Tabrizi SJ (2017) Therapies targeting DNA and RNA in Huntington’s disease. Lancet Neurol 16:837–847. https://doi.org/10.1016/S1474-4422(17)30280-6
Shibata A, Steinlage M, Barton O et al (2017) DNA double-strand break resection occurs during non-homologous end joining in G1 but is distinct from resection during homologous recombination. Mol Cell 65:671–684.e5. https://doi.org/10.1016/j.molcel.2016.12.016
Cho SW, Kim S, Kim Y et al (2014) Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res 24:132–141. https://doi.org/10.1101/gr.162339.113
Ran FA, Hsu PD, Lin CY et al (2013) Double nicking by RNA-guided CRISPR cas9 for enhanced genome editing specificity. Cell 154:1380–1389. https://doi.org/10.1016/j.cell.2013.08.021
Kocher T, Peking P, Klausegger A et al (2017) Cut and paste: efficient homology-directed repair of a dominant negative KRT14 Mutation via CRISPR/Cas9 nickases. Mol Ther 25:2585–2598. https://doi.org/10.1016/j.ymthe.2017.08.015
Eggenschwiler R, Moslem M, Fráguas MS et al (2016) Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase. Nat Publ Gr:1–14. https://doi.org/10.1038/srep38198
Mosbach V, Poggi L, Richard G-F (2019) Trinucleotide repeat instability during double-strand break repair: from mechanisms to gene therapy. Curr Genet 65:17–28. https://doi.org/10.1007/s00294-018-0865-1
Shin JW, Kim K-H, Chao MJ et al (2016) Permanent inactivation of Huntington’s disease mutation by personalized allele-specific CRISPR/Cas9. Hum Mol Genet 25(20):4566–4576. https://doi.org/10.1093/hmg/ddw286
Monteys AM, Ebanks SA, Keiser MS, Davidson BL (2017) CRISPR/Cas9 editing of the mutant Huntingtin Allele in vitro and in vivo. Mol Ther 25:12–23. https://doi.org/10.1016/j.ymthe.2016.11.010
Yang S, Chang R, Yang H et al (2017) CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington’s disease. J Clin Invest 127:2719–2724. https://doi.org/10.1172/JCI92087
Dabrowska M, Juzwa W, Krzyzosiak WJ, Olejniczak M (2018) Precise excision of the CAG tract from the Huntingtin gene by Cas9 nickases. Front Neurosci 12:75. https://doi.org/10.3389/FNINS.2018.00075
Ramlee MK, Yan T, Cheung AMS et al (2015) High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis. Sci Rep 5:15587. https://doi.org/10.1038/srep15587
Haeussler M, Schönig K, Eckert H et al (2016) Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol 17:148. https://doi.org/10.1186/s13059-016-1012-2
Cui Y, Xu J, Cheng M et al (2018) Review of CRISPR/Cas9 sgRNA design tools. Interdiscip Sci Comput Life Sci 10:455–465. https://doi.org/10.1007/s12539-018-0298-z
Yumlu S, Stumm J, Bashir S et al (2017) Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9. Methods 121–122:29–44. https://doi.org/10.1016/j.ymeth.2017.05.009
Dabrowska M, Czubak K, Juzwa W et al (2018) qEva-CRISPR: a method for quantitative evaluation of CRISPR/Cas-mediated genome editing in target and off-target sites. Nucleic Acids Res 46:e101. https://doi.org/10.1093/nar/gky505
Acknowledgments
This work was supported by a grant from the National Science Center (2015/18/E/NZ2/00678) and from the quality-promoting subsidy under the Leading National Research Center (KNOW) program for 2014–2018.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Dabrowska, M., Olejniczak, M. (2020). Gene Therapy for Huntington’s Disease Using Targeted Endonucleases. In: Richard, GF. (eds) Trinucleotide Repeats. Methods in Molecular Biology, vol 2056. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9784-8_17
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9784-8_17
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9783-1
Online ISBN: 978-1-4939-9784-8
eBook Packages: Springer Protocols