RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, Massachusetts 01605, United States.
Howard Hughes Medical Institute, University of Massachusetts Chan Medical School, Worcester, Massachusetts 01605, United States.
ACS Chem Biol. 2022 May 20;17(5):1045-1050. doi: 10.1021/acschembio.2c00116. Epub 2022 Apr 21.
CRISPR-Cas technology has revolutionized genome editing. Its broad and fast-growing application in biomedical research and therapeutics has led to increased demand for guide RNAs. The synthesis of chemically modified single-guide RNAs (sgRNAs) containing >100 nucleotides remains a bottleneck. Here we report the development of a tetrazine ligation method for the preparation of sgRNAs. A tetrazine moiety on the 3'-end of the crRNA and a norbornene moiety on the 5'-end of the tracrRNA enable successful ligation between crRNA and tracrRNA to form sgRNA under mild conditions. Tetrazine-ligated sgRNAs allow efficient genome editing of reporter and endogenous loci in human cells. High-efficiency editing requires structural optimization of the linker.
CRISPR-Cas 技术已经彻底改变了基因组编辑。它在生物医学研究和治疗中的广泛快速应用，导致了对指导 RNA 的需求增加。合成含有 >100 个核苷酸的化学修饰的单指导 RNA（sgRNA）仍然是一个瓶颈。在这里，我们报告了一种用于制备 sgRNA 的四嗪连接方法的开发。crRNA 的 3'-末端上的四嗪部分和 tracrRNA 的 5'-末端上的降冰片烯部分，使得在温和条件下 crRNA 和 tracrRNA 之间能够成功连接，形成 sgRNA。四嗪连接的 sgRNA 允许在人类细胞中有效编辑报告基因和内源性基因座。高效编辑需要对连接子进行结构优化。
