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系统是一类可通过基因敲除（gene knockout, KO）实现特定功能丧失表型构建的强力工具。然而，该方法在原代人类细胞中的应用仍存在较大挑战。
本技术报告报道了一种可在人类脂肪干细胞/祖细胞（adipose stem/progenitor cells, ASCs）基因组中实现功能性基因敲除的可靠实验方案。本研究以Sprouty1（SPRY1）作为CRISPR/Cas9介导基因敲除的模式靶基因，详细阐明了包括CRISPR/Cas9靶序列筛选、适配慢病毒载体的选用在内的完整实验流程，以实现功能性基因敲除。
通过基于PCR的突变检测实验及序列分析，可评估CRISPR/Cas9对SPRY1基因的突变效率。采用RT-qPCR与蛋白质印迹（Western blotting）分别检测其对mRNA及蛋白表达水平的影响。
此外，本研究证实，CRISPR/Cas9介导的SPRY1基因敲除与短发夹RNA（short hairpin RNA, shRNA）介导的基因沉默在消耗Sprouty1蛋白以及抑制成脂分化方面效果相当。
综上，本研究展示了一种可在人类ASCs中实现基因敲除的可靠策略，该方法同样可推广应用于其他原代细胞类型。
<b>缩写：</b>ASC：脂肪干细胞/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：分解分析追踪插入缺失突变（Tracking of indels by decomposition）