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.