Engineering designer beta cells with a CRISPR-Cas9 conjugation platform
Engineering designer beta cells with a CRISPR-Cas9 conjugation platform
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DOI：
10.1038/s41467-020-17725-0
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发表时间：
2020-08-13
影响因子：
16.6
通讯作者：
Choudhary, Amit
中科院分区：
文献类型：
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作者：
Lim, Donghyun;Sreekanth, Vedagopuram;Choudhary, Amit
将蛋白质结构域与Cas9基因融合已经产生了几种变革性技术;然而，遗传修饰仅限于Cas9末端的天然多肽链，这排除了可用于基因编辑的多种分子。在这里，我们报告了化学修饰，允许在Cas9的末端和内部位点上对各种各样的分子进行位点特异性和多位点缀合，为赋予Cas9不同的功能创造了一个平台。使用该平台，可以修饰Cas9以在DNA断裂位点处更精确地掺入外源提供的单链寡核苷酸供体（ssODN）。我们证明ssODN与Cas9的多位点缀合显著提高了精确基因组编辑的效率，并且这样的平台与不同长度的ssODN兼容。通过利用缀合平台，我们成功地改造了INS-1 E，一种β细胞系，以重新利用胰岛素分泌机制，这使得保护性免疫调节因子白细胞介素-10的葡萄糖依赖性分泌成为可能。
Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, the genetic modifications are limited to natural polypeptide chains at the Cas9 termini, which excludes a diverse array of molecules useful for gene editing. Here, we report chemical modifications that allow site-specific and multiple-site conjugation of a wide assortment of molecules on both the termini and internal sites of Cas9, creating a platform for endowing Cas9 with diverse functions. Using this platform, Cas9 can be modified to more precisely incorporate exogenously supplied single-stranded oligonucleotide donor (ssODN) at the DNA break site. We demonstrate that the multiple-site conjugation of ssODN to Cas9 significantly increases the efficiency of precision genome editing, and such a platform is compatible with ssODNs of diverse lengths. By leveraging the conjugation platform, we successfully engineer INS-1E, a beta-cell line, to repurpose the insulin secretion machinery, which enables the glucose-dependent secretion of protective immunomodulatory factor interleukin-10.