Department of Chemistry, Binghamton University, The State University of New York, Binghamton, New York 13902, United States.
ACS Chem Biol. 2022 Mar 18;17(3):509-512. doi: 10.1021/acschembio.1c00900. Epub 2022 Feb 28.
The development of CRISPR-Cas9 mediated gene editing technology is revolutionizing molecular biology, biotechnology, and medicine. However, as with other nucleic acid technologies, CRISPR would greatly benefit from chemical modifications that optimize delivery, activity, and specificity of gene editing. Amide modifications at certain positions of short interfering RNAs have been previously shown to improve their RNAi activity and specificity, which motivated the current study on replacement of selected internucleoside phosphates of CRISPR RNAs with amide linkages. Herein, we show that amide modifications did not interfere with CRISPR-Cas9 activity when placed in the protospacer adjacent motif (PAM) distal region of CRISPR RNAs. In contrast, modification of the seed region led to a loss of DNA cleavage activity at most but not all positions. These results are encouraging for future studies on amides as backbone modifications in CRISPR RNAs.
CRISPR-Cas9 介导的基因编辑技术的发展正在彻底改变分子生物学、生物技术和医学。然而，与其他核酸技术一样，CRISPR 将极大地受益于化学修饰，这些修饰可以优化基因编辑的递呈、活性和特异性。以前已经证明，在短干扰 RNA 的某些位置进行酰胺修饰可以提高其 RNAi 活性和特异性，这促使我们目前对 CRISPR RNA 中选定的核苷间磷酸进行酰胺键替换的研究。在此，我们表明酰胺修饰不会干扰 CRISPR-Cas9 活性，只要将其放置在 CRISPR RNA 的前导序列相邻基序 (PAM) 远端区域。相比之下，修饰种子区域会导致大多数但不是所有位置的 DNA 切割活性丧失。这些结果为未来在 CRISPR RNA 中酰胺作为骨架修饰的研究提供了鼓舞。
