One step engineering of the small-subunit ribosomal RNA using CRISPR/Cas9.
One step engineering of the small-subunit ribosomal RNA using CRISPR/Cas9.
复制标题
DOI：
10.1038/srep30714
复制
发表时间：
2016-08-04
影响因子：
4.6
通讯作者：
Gibson DG
中科院分区：
文献类型：
--
作者：
Kannan K;Tsvetanova B;Chuang RY;Noskov VN;Assad-Garcia N;Ma L;Hutchison Iii CA;Smith HO;Glass JI;Merryman C;Venter JC;Gibson DG
细菌由于其相对简单的基因和基因组结构，在基本分子生物学过程的研究中是不可或缺的。改造细菌染色体的能力对于理解基因功能至关重要。在这里，我们通过结合CRISPR/Cas9系统和酵母重组机制，展示了支原体的小核糖体亚基（16S） RNA的工程。我们在酵母中克隆了mycoides的全基因组，并使用组成型表达的Cas9和体外转录的引导rna来引入工程的16S rRNA基因。通过基因组移植测试工程16S rRNA基因的功能，我们观察到该基因对添加遗传元件或与系统发育上遥远的细菌进行螺旋替换的惊人弹性。虽然该系统可以进一步用于研究16S rRNA的功能，但人们可以设想，“简单”的mycoides基因组可以在此环境中用于研究其他遗传结构和功能，以回答生命的基本问题。
Bacteria are indispensable for the study of fundamental molecular biology processes due to their relatively simple gene and genome architecture. The ability to engineer bacterial chromosomes is quintessential for understanding gene functions. Here we demonstrate the engineering of the small-ribosomal subunit (16S) RNA of Mycoplasma mycoides, by combining the CRISPR/Cas9 system and the yeast recombination machinery. We cloned the entire genome of M. mycoides in yeast and used constitutively expressed Cas9 together with in vitro transcribed guide-RNAs to introduce engineered 16S rRNA genes. By testing the function of the engineered 16S rRNA genes through genome transplantation, we observed surprising resilience of this gene to addition of genetic elements or helix substitutions with phylogenetically-distant bacteria. While this system could be further used to study the function of the 16S rRNA, one could envision the “simple” M. mycoides genome being used in this setting to study other genetic structures and functions to answer fundamental questions of life.