"Split-and-Click" sgRNA.
"Split-and-Click" sgRNA.
复制标题
DOI：
10.1007/978-1-0716-0687-2_5
复制
发表时间：
2021
影响因子：
--
通讯作者：
Lapatrada Taemaitree;Arun Shivalingam;A. El-Sagheer;T. Brown
中科院分区：
文献类型：
--
作者：
Lapatrada Taemaitree;Arun Shivalingam;A. El-Sagheer;T. Brown
CRISPR-Cas9基因编辑依赖于指导Cas9内切核酸酶活性的可编程单向导RNA（sgRNA）。这种RNA通常通过酶促反应产生，然而，随着sgRNA数量的增加，该过程变得耗时，并且不允许掺入可以改善或扩展CRISPR功能的化学修饰。固相RNA合成可以克服这些问题，但是高纯度的全长sgRNA仍然受到当前合成方法的限制。在这里，我们展示了一种“分裂和点击”的方法，将sgRNA分成两个较小的组分-DNA靶向的~20-mer RNA和恒定的Cas9结合的79-mer RNA -并将它们化学连接在一起以产生生物活性的sgRNA。我们的方法的好处在于DNA靶向20-mer的严格纯化，每次需要新的sgRNA时减少恒定79-mer的合成，以及它提供的快速访问定制sgRNA文库。
CRISPR-Cas9 gene editing is dependent on a programmable single guide RNA (sgRNA) that directs Cas9 endonuclease activity. This RNA is often generated by enzymatic reactions, however the process becomes time-consuming as the number of sgRNAs increases and does not allow the incorporation of chemical modifications that can improve or expand the functionality of CRISPR. Solid-phase RNA synthesis can overcome these issues, but highly pure full-length sgRNA remains at the limits of current synthetic methods. Here, we demonstrate a “split-and-click” approach that separates the sgRNA into its two smaller components – a DNA-targeting ~20-mer RNA and a constant Cas9-binding 79-mer RNA – and chemically ligates them together to generate a biologically active sgRNA. The benefits of our approach lie in the stringent purification of the DNA-targeting 20-mer, the reduced synthesis of the constant 79-mer each time a new sgRNA is required, and the rapid access it provides to custom libraries of sgRNAs.