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

摘要

CRISPR / Cas9系统是通过基因敲除（KO）产生特定功能丧失表型的强大工具。然而，这种方法在原代人类细胞中具有挑战性。在此技术报告中，我们提出了一种可靠的协议，可以在人类脂肪干/祖细胞（ASC）的基因组中实现功能性KO。使用Sprouty1（SPRY1）作为CRISPR / Cas9介导的KO的模型靶基因，我们具体说明了该程序，包括CRISPR / Cas9靶序列的选择以及采用合适的慢病毒载体来获得功能性基因KO。CRISPR / Cas9突变SPRY1的效率通过基于PCR的突变检测测定和序列分析确定基因。通过RT-qPCR和蛋白质印迹研究对mRNA和蛋白质水平的影响。另外，我们证明了CRISPR / Cas9介导的SPRY1 KO和shRNA的基因沉默同样有效地消耗Sprouty1蛋白并抑制成脂分化。总之，我们展示了一种在人类ASC中实现基因KO的可靠方法，该方法也可能适用于其他原代细胞类型。缩写： ASC：成脂干/祖细胞；Cas：CRISPR相关系统；CRISPR：成簇的规则间隔回文重复序列；gDNA：基因组DNA；GOI：感兴趣的基因；gRNA：指导RNA；NHEJ：非同源末端连接；插入缺失：在插入可以/德尔行为 PAM：Protospacer相邻主题；sWAT：皮下白色脂肪组织；潮汐：通过分解追踪插入缺失