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.

Knock out aryl hydrocarbon receptor gene in adipose-derived mesenchymal stem cell line (PT-5006) by CRISPR/Cas9

“…These findings were similar to previous study that found a decrease in the expression of AhR and its downstream genes in KO cells utilizing CRISPR/Cas9 technology (Zgarbová & Vrzal, 2022). Disrupting specific genes in stem cells through knockout techniques is a highly effective method for investigating their roles in differentiation, proliferation, and other essential cellular functions (Mandl et al, 2020). Several genome editing methods have been developed to generate KO stem cells, with CRISPR/Cas9 is one of the most powerful systems (Kim et al, 2020;Lee et al, 2022).…”

The circadian transcription factor ARNTL2 is regulated by weight-loss interventions in human white adipose tissue and inhibits adipogenesis

“…Employment of specific shRNAs abolished ARNTL2 mRNA expression but failed to significantly reduce the corresponding protein level (data not shown). A CRISPR/Cas9-mediated gene knockout approach, as recently described by us [58], was ineffective despite the use of several target sequences (data not shown). The failure of both techniques to profoundly reduce the ARNTL2 protein level in ASCs is most likely due to the very high stability of this transcription factor (Supplementary Fig.…”

Distinct Dominant Lineage from In Vitro Expanded Adipose-Derived Stem Cells (ASCs) Exhibits Enhanced Wound Healing Properties

“…Identification of surrogate markers relevant to functionality would therefore go a long way towards developing more effective therapeutic regimes. Efforts have been made to associate RNA or protein signatures with clinically relevant properties [ 20 , 21 , 22 , 23 ], but surface markers provide advantages that defined subpopulations may be purified and investigated [ 12 ]. Some surface markers, mostly CDs studies as a single epitope or in a limited co-expression pattern, have been previously proposed to support certain biological functions early after isolation [ 24 , 25 , 26 ].…”