دورية أكاديمية
Conditional Gene Knockout in Human Cells with Inducible CRISPR/Cas9.
| العنوان: | Conditional Gene Knockout in Human Cells with Inducible CRISPR/Cas9. |
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| المؤلفون: | Snijders KE; Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, UK.; Department of Surgery, University of Cambridge, Cambridge, UK., Cooper JD; Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, UK.; Division of Cardiovascular Medicine, University of Cambridge, Cambridge, UK., Vallier L; Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, UK. lv225@cam.ac.uk.; Department of Surgery, University of Cambridge, Cambridge, UK. lv225@cam.ac.uk.; Wellcome Trust Sanger Institute, Hinxton, UK. lv225@cam.ac.uk., Bertero A; Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, UK. abertero@uw.edu.; Department of Surgery, University of Cambridge, Cambridge, UK. abertero@uw.edu.; Department of Pathology, University of Washington, Seattle, WA, USA. abertero@uw.edu. |
| المصدر: | Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2019; Vol. 1961, pp. 185-209. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't |
| اللغة: | English |
| بيانات الدورية: | Publisher: Humana Press Country of Publication: United States NLM ID: 9214969 Publication Model: Print Cited Medium: Internet ISSN: 1940-6029 (Electronic) Linking ISSN: 10643745 NLM ISO Abbreviation: Methods Mol Biol |
| أسماء مطبوعة: | Publication: Totowa, NJ : Humana Press Original Publication: Clifton, N.J. : Humana Press, |
| مواضيع طبية MeSH: | CRISPR-Cas Systems/*genetics , Gene Knockout Techniques/*methods, Humans ; Pluripotent Stem Cells/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics |
| مستخلص: | The advent of the easily programmable and efficient CRISPR/Cas9 nuclease system has revolutionized genetic engineering. While conventional gene knockout experiments using CRISPR/Cas9 are very valuable, these are not well suited to study stage-specific gene function in dynamic situations such as development or disease. Here we describe a CRISPR/Cas9-based OPTimized inducible gene KnockOut method (OPTiKO) for conditional loss-of-function studies in human cells. This approach relies on an improved tetracycline-inducible system for conditional expression of single guide RNAs (sgRNAs) that drive Cas9 activity. In order to ensure homogeneous and stable expression, the necessary transgenes are expressed following rapid and efficient single-step genetic engineering of the AAVS1 genomic safe harbor. When implemented in human pluripotent stem cells (hPSCs), the approach can be then efficiently applied to virtually any hPSC-derived human cell type at various stages of development or disease. |
| معلومات مُعتمدة: | FS/11/77/39327 United Kingdom BHF_ British Heart Foundation; United Kingdom WT_ Wellcome Trust; PSAG028 United Kingdom MRC_ Medical Research Council; United Kingdom DH_ Department of Health; MC_PC_12009 United Kingdom MRC_ Medical Research Council; G0701448 United Kingdom MRC_ Medical Research Council |
| فهرسة مساهمة: | Keywords: AAVS1; CRISPR/Cas9; Human pluripotent stem cells; Inducible knockout; Tetracycline; sgRNA |
| المشرفين على المادة: | 0 (RNA, Guide, CRISPR-Cas Systems) |
| تواريخ الأحداث: | Date Created: 20190327 Date Completed: 20190726 Latest Revision: 20240104 |
| رمز التحديث: | 20240104 |
| DOI: | 10.1007/978-1-4939-9170-9_12 |
| PMID: | 30912047 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 1940-6029 |
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| DOI: | 10.1007/978-1-4939-9170-9_12 |