Phosphonoacetate Modifications Enhance the Stability and Editing Yields of Guide RNAs for Cas9 Editors

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′-O-methyl-3′-phosphonoacetate
(MP) modifications can be substantially more effective than 2′-O-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（CRISPR gene editing）与控制系统持续取得新进展，并催生了诸多新颖的研究方向与临床应用。针对CRISPR系统的性能优化手段——如优化其在细胞、组织及生物体中的活性持续时长、限制脱靶活性——对拓展基于CRISPR的系统的应用范围具有至关重要的意义。通过探究向导RNA（guide RNAs, gRNAs）在特定位置与组合模式下的各类化学修饰效果，本研究发现，在单向导RNA（single-guide RNAs, sgRNAs）的3'端引入2′-O-甲基-3′-膦酸乙酸酯（2′-O-methyl-3′-phosphonoacetate, MP）修饰，其效果显著优于2′-O-甲基-3′-硫代磷酸酯（2′-O-methyl-3′-phosphorothioate, MS）修饰，可有效提升基因编辑产率；在部分实验中，人类细胞内的编辑效率可提升一个数量级。MP修饰的3'端在与Cas信使RNA（messenger RNA, mRNA）共转染的向导RNA中表现尤为突出，因向导RNA需在细胞内持续存留直至Cas蛋白完成表达。我们验证了该MP增强效应在与BE4信使RNA共转染、用于胞嘧啶碱基编辑的单向导RNA中同样成立；同时证实，在与PE2信使RNA共转染的先导编辑向导RNA（prime editing guide RNAs, pegRNAs）的3'端引入MP修饰，可实现最优的先导编辑效率。在血清存在的条件下，与Cas9信使RNA共同递送的、带有MP修饰3'端的单向导RNA，其编辑效率相较带有MS修饰3'端的单向导RNA有显著提升；而在增强转染的核糖核蛋白（ribonucleoprotein, RNP）复合物活性方面，MP修饰的提升效果则相对温和。本研究结果表明，MP可作为合成向导RNA 3'端的性能增强修饰手段，尤其适用于向导RNA易受核酸外切酶介导降解的应用场景。