Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch.
Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch.
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工程热点分析确定了变构 CRISPR-Cas9 开关。
DOI：
10.1038/nbt.3528
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发表时间：
2016-06
影响因子：
46.9
通讯作者：
Savage DF
中科院分区：
文献类型：
--
作者：
Oakes BL;Nadler DC;Flamholz A;Fellmann C;Staahl BT;Doudna JA;Savage DF
来自化脓性链球菌的CRISPR相关蛋白Cas9是一种RNA引导的DNA内切核酸酶，广泛用于基因组修饰。然而，限制Cas9工程化的结构约束尚未确定。在这里，我们使用随机插入诱变对Cas9进行了实验性分析，并描绘了能够耐受PDZ结构域插入而不破坏酶的结合和切割功能的结构中的热点。正交结构域或结构域的组合可以以最小的功能后果插入到所鉴定的位点中。为了说明所鉴定位点的效用，我们通过插入雌激素受体α配体结合结构域构建了变构调节的Cas9。这种蛋白质在原核和真核细胞中显示出稳健的配体依赖性激活，建立了用于诱导型和可逆的Cas9激活的通用单组分系统。因此，结构域插入分析促进了新的Cas9功能的快速产生，并为Cas9的未来工程化提供了有用的数据。
The CRISPR-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disrupting the enzyme’s binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the Estrogen Receptor α Ligand Binding Domain. This protein displayed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9.