Agilent Research Laboratories, Santa Clara, California 95051, United States.
Agilent Research Laboratories, Boulder, Colorado 80303, United States.
Biochemistry. 2023 Dec 19;62(24):3512-3520. doi: 10.1021/acs.biochem.1c00768. Epub 2022 Apr 18.
CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the duration of activity in cells, tissues, and organisms, as well as limiting off-target activities, have been extremely important for expanding the utility of CRISPR-based systems. By investigating the effects of various chemical modifications in guide RNAs (gRNAs) at defined positions and combinations, we find that 2'--methyl-3'-phosphonoacetate (MP) modifications can be substantially more effective than 2'--methyl-3'-phosphorothioate (MS) modifications at the 3' ends of single-guide RNAs (sgRNAs) to promote high editing yields, in some instances showing an order of magnitude higher editing yield in human cells. MP-modified 3' ends are especially effective at promoting the activity of guide RNAs cotransfected with Cas messenger RNA (mRNA), as the gRNA must persist in cells until the Cas protein is expressed. We demonstrate such an MP enhancement for sgRNAs cotransfected with a BE4 mRNA for cytidine base editing and also demonstrate that MP at the 3' ends of prime editing guide RNAs (pegRNAs) cotransfected with PE2 mRNA can promote maximal prime editing yields. In the presence of serum, sgRNAs with MP-modified 3' ends showed marked improvements in editing efficiency over sgRNAs with MS-modified 3' ends codelivered with Cas9 mRNA and showed more modest improvements at enhancing the activity of transfected ribonucleoprotein (RNP) complexes. Our results suggest that MP should be considered as a performance-enhancing modification for the 3' ends of synthetic gRNAs, especially in situations where the guide RNAs may be susceptible to exonuclease-mediated degradation.
CRISPR 基因编辑和控制系统不断涌现，并激发了新的研究和临床应用。优化 CRISPR 在细胞、组织和生物体中的活性持续时间以及限制脱靶活性等方面的进展，对于扩展基于 CRISPR 的系统的实用性非常重要。通过研究在定义位置和组合的向导 RNA（gRNA）中各种化学修饰的影响，我们发现 2'-O-甲基-3'-磷酸乙酰基（MP）修饰在单向导 RNA（sgRNA）的 3'末端比 2'-O-甲基-3'-硫代磷酸酯（MS）修饰更有效，以提高编辑效率，在某些情况下，在人类细胞中显示出数量级更高的编辑效率。MP 修饰的 3'末端特别有效地促进与 Cas 信使 RNA（mRNA）共转染的向导 RNA 的活性，因为 gRNA 必须在细胞中持续存在，直到表达 Cas 蛋白。我们证明了这种 MP 增强作用对于与 BE4 mRNA 共转染的 sgRNA 用于胞嘧啶碱基编辑，并且还证明了与 PE2 mRNA 共转染的 Prime 编辑向导 RNA（pegRNA）的 3'末端的 MP 可以促进最大的 Prime 编辑产量。在存在血清的情况下，具有 MP 修饰的 3'末端的 sgRNA 与 Cas9 mRNA 共转染的具有 MS 修饰的 3'末端的 sgRNA 相比，编辑效率有明显提高，并在增强转染核糖核蛋白（RNP）复合物的活性方面有适度提高。我们的结果表明，MP 应该被视为合成 gRNA 的 3'末端的性能增强修饰，特别是在向导 RNA 可能易受核酸外切酶介导的降解的情况下。
