Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9
Year: 2018
Type: article
Abstract: 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.
DOI: 10.3791/57278
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: bronze
Funders: Cancer Research Institute, Cancer Prevention and Research Institute of Texas, Edward P. Evans Foundation, Gabrielle's Angel Foundation for Cancer Research, Evans Medical Foundation, National Institutes of Health, National Cancer Institute, National Institute of Allergy and Infectious Diseases, National Institute of Diabetes and Digestive and Kidney Diseases, National Center for Research Resources
Awards: Mesenchymal Stromal Cells Regulate Hematopoietic Stem Cell Aging, BD Biosciences Special Order LSRII, G2408992657, Role of DNA METHYLTRANSFERASE 3A in Hematologic Malignancies, G3783386658, G5465708471, G6222068007, REGULATION OF HEMATOPOIETIC PROGENITORS BY DE NOVO DNA METHYLATION, Recruitment of First-Time, Tenure-Track Faculty Members, G6962160970, G7837447426, G8059554933, Baylor College of Medicine Comprehensive Cancer Training Program, G9000184390, G9000198215, G9000229161, G9000240210, DNMT3A in Development of Hematologic Malignancies, Baylor College of Medicine Cancer Center - Cancer Center Support Grant, SPORE in Lymphoma
OpenAlex ID: W4205982915