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Genome-wide CRISPR/Cas9-knockout in human induced Pluripotent Stem Cell (iPSC)-derived macrophages.
Navarro-Guerrero, Elena; Tay, Chwen; Whalley, Justin P; Cowley, Sally A; Davies, Ben; Knight, Julian C; Ebner, Daniel.
Afiliación
  • Navarro-Guerrero E; Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Oxford, UK.
  • Tay C; Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.
  • Whalley JP; Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.
  • Cowley SA; James Martin Stem Cell Facility, Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.
  • Davies B; Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.
  • Knight JC; Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK. julian@well.ox.ac.uk.
  • Ebner D; Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Oxford, UK. daniel.ebner@ndm.ox.ac.uk.
Sci Rep ; 11(1): 4245, 2021 02 19.
Article en En | MEDLINE | ID: mdl-33608581
Genome engineering using CRISPR/Cas9 technology enables simple, efficient and precise genomic modifications in human cells. Conventional immortalized cell lines can be easily edited or screened using genome-wide libraries with lentiviral transduction. However, cell types derived from the differentiation of induced Pluripotent Stem Cells (iPSC), which often represent more relevant, patient-derived models for human pathology, are much more difficult to engineer as CRISPR/Cas9 delivery to these differentiated cells can be inefficient and toxic. Here, we present an efficient, lentiviral transduction protocol for delivery of CRISPR/Cas9 to macrophages derived from human iPSC with efficiencies close to 100%. We demonstrate CRISPR/Cas9 knockouts for three nonessential proof-of-concept genes-HPRT1, PPIB and CDK4. We then scale the protocol and validate for a genome-wide pooled CRISPR/Cas9 loss-of-function screen. This methodology enables, for the first time, systematic exploration of macrophage involvement in immune responses, chronic inflammation, neurodegenerative diseases and cancer progression, using efficient genome editing techniques.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Diferenciación Celular / Células Madre Pluripotentes Inducidas / Sistemas CRISPR-Cas / Macrófagos Límite: Humans Idioma: En Revista: Sci Rep Año: 2021 Tipo del documento: Article Pais de publicación: Reino Unido
Texto completo
Añadir a Mi BVS
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XML
PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Diferenciación Celular / Células Madre Pluripotentes Inducidas / Sistemas CRISPR-Cas / Macrófagos Límite: Humans Idioma: En Revista: Sci Rep Año: 2021 Tipo del documento: Article Pais de publicación: Reino Unido