Obesity and type 2 diabetes (T2D) are associated with poor tissue responses to insulin [1,2], disturbances in glucose and lipid fluxes [3-5] and comorbidities including steatohepatitis [6] and cardiovascular disease [7,8]. Despite extensive efforts at prevention and treatment [9,10], diabetes afflicts over 400 million people worldwide [11]. Whole body metabolism is regulated by adipose tissue depots [12-14], which include both lipid-storing white adipocytes and less abundant 'brown' and 'brite/beige' adipocytes that express thermogenic uncoupling protein UCP1 and secrete factors favorable to metabolic health [15-18]. Application of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing [19,20] to enhance 'browning' of white adipose tissue is an attractive therapeutic approach to T2D. However, the problems of cell-selective delivery, immunogenicity of CRISPR reagents and long term stability of the modified adipocytes are formidable. To overcome these issues, we developed methods that deliver complexes of SpyCas9 protein and sgRNA ex vivo to disrupt the thermogenesis suppressor gene NRIP1 [21,22] with near 100% efficiency in human or mouse adipocytes. NRIP1 gene disruption at discrete loci strongly ablated NRIP1 protein and upregulated expression of UCP1 and beneficial secreted factors, while residual Cas9 protein and sgRNA were rapidly degraded. Implantation of the CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreased adiposity and liver triglycerides while enhancing glucose tolerance compared to mice implanted with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic modification of human adipocytes without exposure of the recipient to immunogenic Cas9 or delivery vectors.

中文翻译：

---

CRISPR增强人类脂肪细胞“变褐”作为代谢疾病的细胞疗法

肥胖和2型糖尿病（T2D）与组织对胰岛素的不良反应[1,2]，葡萄糖和脂质通量的紊乱[3-5]和合并症包括脂肪性肝炎[6]和心血管疾病[7,8]相关。尽管在预防和治疗方面付出了巨大的努力[9,10]，但全世界仍有4亿人患有糖尿病[11]。全身代谢受脂肪组织贮库的调控[12-14]，既包括储脂的白色脂肪细胞，又包括表达生热解偶联蛋白UCP1和有利于代谢健康的分泌因子的数量较少的“棕色”和“棕/米色”脂肪细胞[ 15-18]。应用簇状规则间隔的短回文重复序列（CRISPR）基因编辑[19,20]来增强白色脂肪组织的“褐变”是一种吸引人的T2D治疗方法。然而，细胞选择性传递，CRISPR试剂的免疫原性和修饰的脂肪细胞的长期稳定性等问题令人生畏。为了克服这些问题，我们开发了一种方法，可以在体外或体外将SpyCas9蛋白和sgRNA的复合物破坏热生成抑制基因NRIP1 [21,22]，在人类或小鼠脂肪细胞中的效率接近100％。离散位点处的NRIP1基因破坏强烈消融了NRIP1蛋白，并上调了UCP1和有益分泌因子的表达，而残留的Cas9蛋白和sgRNA则迅速降解。与植入未经修饰的脂肪细胞的小鼠相比，将CRISPR增强的人类或小鼠褐色样脂肪细胞植入高脂饮食喂养的小鼠可降低脂肪和肝脏甘油三酸酯，同时提高葡萄糖耐量。