| Autor: |
Sheikh MA; Department of Biochemistry and Molecular Biology, College of Medicine and Health Sciences, United Arab Emirates University., Afandi FH; Department of Biochemistry and Molecular Biology, College of Medicine and Health Sciences, United Arab Emirates University., Iannello G; Columbia Stem Cell Initiative, Stem Cell Core, Columbia University Irving Medical Center., Corneo B; Columbia Stem Cell Initiative, Stem Cell Core, Columbia University Irving Medical Center., Emerald BS; Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University; Zayed Center for Health Sciences, United Arab Emirates University; ASPIRE Precision Medicine Research Institute Abu Dhabi (PMRI-AD), United Arab Emirates University., Ansari SA; Department of Biochemistry and Molecular Biology, College of Medicine and Health Sciences, United Arab Emirates University; Zayed Center for Health Sciences, United Arab Emirates University; ASPIRE Precision Medicine Research Institute Abu Dhabi (PMRI-AD), United Arab Emirates University; sansari@uaeu.ac.ae. |
| Jazyk: |
angličtina |
| Zdroj: |
Journal of visualized experiments : JoVE [J Vis Exp] 2024 Nov 01 (213). Date of Electronic Publication: 2024 Nov 01. |
| DOI: |
10.3791/67296 |
| Abstrakt: |
The CRISPR-Cas9 system for genome editing has revolutionized gene function studies in mammalian cells, including stem cells. However, the practical application of this technique, particularly in pluripotent stem cells, presents certain challenges, such as being time- and labor-intensive and having low editing efficiency. Here, we describe the generation of a CRISPR-mediated gene knockout in a human embryonic stem cell (hESC) line stably expressing sgRNAs for the L2HGDH gene, using a highly efficient and stable lentiviral-mediated gene delivery system. The sgRNAs targeting exon 1 of the L2HGDH gene were chemically synthesized and cloned into the lentiCRISPR v2-puro vector, which combines the constitutive expression of sgRNAs with Cas9 in a highly efficient single-vector system to achieve higher lentiviral titers for hESC infection and stable selection using puromycin. Puromycin-selected cells were further expanded, and single-cell clones were obtained using the limited dilution method. The single clones were expanded, and several homozygous knockout clones for the L2HGDH gene were obtained, as confirmed by a 100% reduction in L2HGDH expression using Western blot analysis. Furthermore, using MSBSP-PCR, the CRISPR mutation site was mapped upstream of the PAM recognition sequence of Cas9 in the selected homozygous clones. Sanger sequencing was performed to analyze the exact insertions/deletions, and functional characterization of the clones was conducted. This method produced a significantly higher percentage of homozygous deletions compared to previously reported non-viral gene delivery methods. Although this report focuses on the L2HGDH gene, this robust and cost-effective approach can be used to create homozygous knockouts for other genes in pluripotent stem cells for gene function studies. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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