دورية أكاديمية
Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9.
| العنوان: | Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9. |
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| المؤلفون: | Brunetti L; Stem Cells & Regenerative Medicine Center, Baylor College of Medicine; Center for Cell and Gene Therapy, Baylor College of Medicine; Centro di Ricerca Emato-Oncologica (CREO), University of Perugia., Gundry MC; Stem Cells & Regenerative Medicine Center, Baylor College of Medicine; Center for Cell and Gene Therapy, Baylor College of Medicine; Department of Molecular & Human Genetics, Baylor College of Medicine., Kitano A; Department of Molecular & Human Genetics, Baylor College of Medicine., Nakada D; Stem Cells & Regenerative Medicine Center, Baylor College of Medicine; Center for Cell and Gene Therapy, Baylor College of Medicine; Department of Molecular & Human Genetics, Baylor College of Medicine., Goodell MA; Stem Cells & Regenerative Medicine Center, Baylor College of Medicine; Center for Cell and Gene Therapy, Baylor College of Medicine; Department of Molecular & Human Genetics, Baylor College of Medicine; Texas Children's Hospital & Houston Methodist Hospital; goodell@bcm.edu. |
| المصدر: | Journal of visualized experiments : JoVE [J Vis Exp] 2018 Apr 10 (134). Date of Electronic Publication: 2018 Apr 10. |
| نوع المنشور: | Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Video-Audio Media |
| اللغة: | English |
| بيانات الدورية: | Publisher: MYJoVE Corporation Country of Publication: United States NLM ID: 101313252 Publication Model: Electronic Cited Medium: Internet ISSN: 1940-087X (Electronic) Linking ISSN: 1940087X NLM ISO Abbreviation: J Vis Exp Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: [Boston, Mass. : MYJoVE Corporation, 2006]- |
| مواضيع طبية MeSH: | CRISPR-Cas Systems/*genetics , Hematopoietic Stem Cells/*metabolism , Stem Cells/*metabolism, Animals ; Gene Editing ; Humans ; Mice |
| مستخلص: | Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols. A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs. |
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| معلومات مُعتمدة: | R01 DK107413 United States DK NIDDK NIH HHS; S10 RR024574 United States RR NCRR NIH HHS; P30 CA125123 United States CA NCI NIH HHS; P50 CA126752 United States CA NCI NIH HHS; R01 CA183252 United States CA NCI NIH HHS; R01 DK092883 United States DK NIDDK NIH HHS |
| تواريخ الأحداث: | Date Created: 20180501 Date Completed: 20180723 Latest Revision: 20190329 |
| رمز التحديث: | 20240628 |
| مُعرف محوري في PubMed: | PMC5933422 |
| DOI: | 10.3791/57278 |
| PMID: | 29708546 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 1940-087X |
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| DOI: | 10.3791/57278 |