Abstract
Genetic information transferred by HIV-1-based lentiviral vectors as single-stranded RNA is converted to double-stranded DNA by reverse transcription and subsequently inserted into the genome of recipient cells. Integration into the genome allows stable, long-term expression of genes-of-interest driven by promoter sequences contained within the vector. This technology can be used as a standard method for production of cells stably expressing Cas9 protein and single guide RNA (sgRNA), the key components of the CRISPR genome editing system. Here, we provide a protocol for production and validation of VSV-G-pseudotyped lentiviral vectors for delivery of the CRISPR system and generation of knockout cell lines.
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Acknowledgments
The authors are grateful to Feng Zhang and colleagues for making lentiviral vector plasmids available through Addgene. JGM and members of the JGM lab are grateful for all support provided to projects involving lentiviral vector technologies, including grants from the Danish Council for Independent Research │ Medical Sciences (grant DFF-4004-00220), The Lundbeck Foundation (grant R126-2012-12456), the Novo Nordisk Foundation, Aase og Ejnar Danielsens Fond, Agnes og Poul Friis Fond, Carl og Ellen Hertz’ legat til læge- og naturvidenskaben, Harboefonden, Arvid Nilssons Fond, Fonden af 1870, Else og Mogens Wedell-Wedellsborgs Fond, Einar Willumsens Mindelegat, Emil C. Hertz og hustru Inger Hertz’s Fond, Holger Hjortenberg og Hustru Dagmar Hjortenbergs Fond, Krista og Viggo Petersens Fond, and Andersen-Isted Fonden.
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Ryø, L.B., Thomsen, E.A., Mikkelsen, J.G. (2019). Production and Validation of Lentiviral Vectors for CRISPR/Cas9 Delivery. In: Luo, Y. (eds) CRISPR Gene Editing. Methods in Molecular Biology, vol 1961. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9170-9_7
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DOI: https://doi.org/10.1007/978-1-4939-9170-9_7
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