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CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells.

ABSTRACT: The CRISPR/Cas9 system is a powerful tool to generate a specific loss-of-function phenotype by gene knockout (KO). However, this approach is challenging in primary human cells. In this technical report, we present a reliable protocol to achieve a functional KO in the genome of human adipose stem/progenitor cells (ASCs). Using Sprouty1 (SPRY1) as a model target gene for a CRISPR/Cas9 mediated KO, we particularize the procedure including the selection of the CRISPR/Cas9 target sequences and the employment of appropriate lentiviral vectors to obtain a functional gene KO. The efficiency of CRISPR/Cas9 to mutate the SPRY1 gene is determined by a PCR-based mutation detection assay and sequence analysis. Effects on mRNA and protein levels are studied by RT-qPCR and Western blotting. In addition, we demonstrate that CRISPR/Cas9 mediated SPRY1 KO and gene silencing by shRNA are similarly effective to deplete the Sprouty1 protein and to inhibit adipogenic differentiation. In summary, we show a reliable approach to achieve a gene KO in human ASCs, which could also apply to other primary cell types. Abbreviations: ASC: Adipogenic Stem/Progenitor Cell; Cas: CRISPR-associated system; CRISPR: Clustered Regularly Interspaced Palindromic Repeat; gDNA: Genomic DNA; GOI: Gene of interest; gRNA: Guide RNA; NHEJ: Non-homologous end joining; Indel: Insertion/Deletion; PAM: Protospacer adjacent motif; sWAT: Subcutaneous white adipose tissue; TIDE: Tracking of indels by decomposition.

SUBMITTER: Mandl M

PROVIDER: S-EPMC7575003 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells.

Mandl Markus M Ritthammer Heike H Ejaz Asim A Wagner Sonja A SA Hatzmann Florian M FM Baumgarten Saphira S Viertler Hans P HP Zwierzina Marit E ME Mattesich Monika M Schiller Valerie V Rauchenwald Tina T Ploner Christian C Waldegger Petra P Pierer Gerhard G Zwerschke Werner W

Adipocyte 20201201 1

The CRISPR/Cas9 system is a powerful tool to generate a specific loss-of-function phenotype by gene knockout (KO). However, this approach is challenging in primary human cells. In this technical report, we present a reliable protocol to achieve a functional KO in the genome of human adipose stem/progenitor cells (ASCs). Using Sprouty1 (<i>SPRY1</i>) as a model target gene for a CRISPR/Cas9 mediated KO, we particularize the procedure including the selection of the CRISPR/Cas9 target sequences and ...[more]

PMID: 33070670

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Project description:Xeroderma pigmentosum group C (XPC) is a versatile protein crucial for sensing DNA damage in the global genome nucleotide excision repair (GG-NER) pathway. This pathway is vital for mammalian cells, acting as their essential approach for repairing DNA lesions stemming from interactions with environmental factors, such as exposure to ultraviolet (UV) radiation from the sun. Loss-of-function mutations in the XPC gene confer a photosensitive phenotype in XP-C patients, resulting in the accumulation of unrepaired UV-induced DNA damage. This remarkable increase in DNA damage tends to elevate by 10,000-fold the risk of developing melanoma and non-melanoma skin cancers. To date, creating accurate and reproducible models to study human XP-C disease has been an important challenge. To tackle this, we used CRISPR-Cas9 technology in order to knockout the XPC gene in various human skin cells (keratinocytes, fibroblasts, and melanocytes). After validation of the knockout in these edited skin cells, we showed that they recapitulate the major phenotypes of XPC mutations: photosensitivity and the impairment of UV-induced DNA damage repair. Moreover, these knockout cells demonstrated a reduced proliferative capacity compared to their respective controls. Finally, to better mimic the disease environment, we built a 3D reconstructed skin using these XPC knockout skin cells. This model exhibited an abnormal behavior, showing an extensive remodeling of its extracellular matrix compared to normal skin. Analyzing the composition of the fibroblast secretome revealed a significant augmented shift in the inflammatory response following XPC knockout. Our innovative "disease on a dish" approach can provide valuable insights into the molecular mechanisms underlying XP-C disease, paving the way to design novel preventive and therapeutic strategies to alleviate the disease phenotype. Also, given the high risk of skin cancer onset in XP-C disease, our new approach can serve as a link to draw novel insights into this elusive field.

Project description:BackgroundThe tumor microenvironment within the breast is rich in adipose elements. The interaction between adipose cells and breast cancer is poorly understood, particularly as it pertains to patients with genetic susceptibility to breast cancer. This study focuses on the phenotype of human adipose-derived stem cells with the BRCA1 mutation and the effect they may have on breast cancer cell behavior.MethodsCRISPR/Cas9 was used to generate de novo BRCA1-knockdown human adipose-derived stem cells. The effect of the BRCA1 knockdown on the adipose-derived stem cell phenotype was compared to wild-type adipose-derived stem cells and patient-derived breast adipose-derived stem cells with known BRCA1 mutations. Interactions between adipose-derived stem cells and the MDA-MB-231 breast cancer cell line were evaluated.ResultsBRCA1-knockdown adipose-derived stem cells stimulated MDA-MB-231 proliferation (1.4-fold increase on day 4; p = 0.0074) and invasion (2.3-fold increase on day 2; p = 0.0171) compared to wild-type cells. Immunofluorescence staining revealed higher levels of phosphorylated ataxia telangiectasia-mutated activation in BRCA1-knockdown cells (72.9 ± 5.32 percent versus 42.9 ± 4.97 percent; p = 0.0147), indicating higher levels of DNA damage. Beta-galactosidase staining demonstrated a significantly higher level of senescence in BRCA1-knockdown cells compared with wild-type cells (7.9 ± 0.25 percent versus 0.17 ± 0.17 percent; p < 0.0001). Using quantitative enzyme-linked immunosorbent assay to evaluate conditioned media, the authors found significantly higher levels of interleukin-8 in BRCA1-knockdown cells (2.57 ± 0.32-fold; p = 0.0049).ConclusionsThe authors show for the first time that the BRCA1 mutation affects the adipose-derived stem cell phenotype. Moreover, CRISPR/Cas9-generated BRCA1-knockdown adipose-derived stem cells stimulate a more aggressive behavior in breast cancer cells than wild-type adipose-derived stem cells. This appears to be related to increased inflammatory cytokine production by means of a DNA damage-mediated cell senescence pathway.

Project description:BackgroundThe application of CRISPR/Cas9 technology in human induced pluripotent stem cells (hiPSC) holds tremendous potential for basic research and cell-based gene therapy. However, the fulfillment of these promises relies on the capacity to efficiently deliver exogenous nucleic acids and harness the repair mechanisms induced by the nuclease activity in order to knock-out or repair targeted genes. Moreover, transient delivery should be preferred to avoid persistent nuclease activity and to decrease the risk of off-target events. We recently developed bacteriophage-chimeric retrovirus-like particles that exploit the properties of bacteriophage coat proteins to package exogenous RNA, and the benefits of lentiviral transduction to achieve highly efficient, non-integrative RNA delivery in human cells. Here, we investigated the potential of bacteriophage-chimeric retrovirus-like particles for the non-integrative delivery of RNA molecules in hiPSC for CRISPR/Cas9 applications.ResultsWe found that these particles efficiently convey RNA molecules for transient expression in hiPSC, with minimal toxicity and without affecting the cell pluripotency and subsequent differentiation. We then used this system to transiently deliver in a single step the CRISPR-Cas9 components (Cas9 mRNA and sgRNA) to generate gene knockout with high indel rate (up to 85%) at multiple loci. Strikingly, when using an allele-specific sgRNA at a locus harboring compound heterozygous mutations, the targeted allele was not altered by NHEJ/MMEJ, but was repaired at high frequency using the homologous wild type allele, i.e., by interallelic gene conversion.ConclusionsOur results highlight the potential of bacteriophage-chimeric retrovirus-like particles to efficiently and safely deliver RNA molecules in hiPSC, and describe for the first time genome engineering by gene conversion in hiPSC. Harnessing this DNA repair mechanism could facilitate the therapeutic correction of human genetic disorders in hiPSC.

Dataset Information

CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells.

Publications

CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells.

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