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.
journal_name
Adipocytejournal_title
Adipocyteauthors
Mandl M,Ritthammer H,Ejaz A,Wagner SA,Hatzmann FM,Baumgarten S,Viertler HP,Zwierzina ME,Mattesich M,Schiller V,Rauchenwald T,Ploner C,Waldegger P,Pierer G,Zwerschke Wdoi
10.1080/21623945.2020.1834230subject
Has Abstractpub_date
2020-12-01 00:00:00pages
626-635issue
1eissn
2162-3945issn
2162-397Xjournal_volume
9pub_type
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