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

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 the employment of appropriate lentiviral vectors to obtain a functional gene KO. The efficiency of CRISPR/Cas9 to mutate the <i>SPRY1</i> 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 <i>SPRY1</i> 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. <b>Abbreviations:</b> 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

CRISPR/Cas9系统是一种通过基因敲除（KO）实现特定功能丧失表型的强力工具，但该方法在原代人类细胞中应用难度较大。在本技术报告中，我们报道了一种可在人类脂肪组织干细胞/祖细胞（adipose stem/progenitor cells，简称ASCs）基因组中实现功能性基因敲除的可靠方案。
以Sprouty1（SPRY1）作为CRISPR/Cas9介导基因敲除的模式靶基因，我们详细阐述了完整操作流程，包括CRISPR/Cas9靶序列的筛选以及合适慢病毒载体的使用，以达成功能性基因敲除。
我们通过基于PCR的突变检测实验与序列分析，对CRISPR/Cas9介导SPRY1基因突变的效率进行验证；并利用逆转录实时定量聚合酶链反应（RT-qPCR）与蛋白质印迹法（Western blotting），研究其对靶基因mRNA与蛋白质水平的影响。
此外，我们证实CRISPR/Cas9介导的SPRY1基因敲除与短发夹RNA（short hairpin RNA，简称shRNA）介导的基因沉默在耗尽Sprouty1蛋白以及抑制成脂分化方面效果相当。
综上，我们展示了一种可在人类ASCs中实现基因敲除的可靠方法，该方案同样可推广至其他原代细胞类型。
**缩写说明：** ASC：成脂干细胞/祖细胞（Adipogenic Stem/Progenitor Cell）；Cas：CRISPR相关系统（CRISPR-associated system）；CRISPR：成簇规律间隔短回文重复序列（Clustered Regularly Interspaced Palindromic Repeat）；gDNA：基因组DNA（Genomic DNA）；GOI：目的基因（Gene of interest）；gRNA：向导RNA（Guide RNA）；NHEJ：非同源末端连接（Non-homologous end joining）；Indel：插入/缺失突变（Insertion/Deletion）；PAM：原间隔序列邻近基序（Protospacer adjacent motif）；sWAT：皮下白色脂肪组织（Subcutaneous white adipose tissue）；TIDE：分解法检测插入缺失突变（Tracking of indels by decomposition）