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

Abstract

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 offtarget 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 exonucleasemediated degradation.

Treatment of a genetic liver disease in mice through transient prime editor expression

“…This pegRNA has previously been successfully utilized for in vivo prime editing in the liver using AAV and AdV mediated delivery 19 . To protect pegRNAs from exonucleases they were modified with 2′-O-methyl-3′-phosphorothioate (MS) at the 5’ end and 2′-O-methyl-3′-phosphonoacetate (MP) at the 3’ end 39 . In addition, we generated pegRNAs in which the RTT was substituted with deoxyribose nucleotides to further protect it from degradation by endonucleases (DNA-mod pegRNAs) ( Fig.…”

Delivering the CRISPR/Cas9 system for engineering gene therapies: Recent cargo and delivery approaches for clinical translation

“…Frontiers in Bioengineering and Biotechnology frontiersin.org thus increasing the sgRNA selectively (Figure 1B) (Ryan et al, 2022). While these data are promising, such chemically modified nucleic acid sequences may induce unwanted and unpredictable immunological responses.…”

“…(B) Specific chemical modifications (left) to nucleotides on both the 5′ and 3′ ends of synthesized sgRNA have increased on-target and off-target gene disruption in primary human T cells for the HBB gene (right). Image reproduced from ref ( Ryan et al, 2022 ) with permission from American Chemical Society. License CC BY-NC-ND 4.0 https://creativecommons.org/licenses/by-nc-nd/4.0/ .…”

“… Jing et al (2021) included the same MS modification to their sgRNA and observed a 15% higher knock-in efficiency compared to unmodified sgRNA when attempting to knock-in predetermined template plasmids into primary human T cells. Ryan et al (2022) similarly engineered the MS and MP nucleotide modifications to their synthesized sgRNA structures and observed increased indel editing for the HBB gene within human primary T cells. By incorporating these alternations within the internal structure of these molecules, they were able to decrease the percentage of off-target indels thus increasing the sgRNA selectively ( Figure 1B ) ( Ryan et al, 2022 ).…”

“… Ryan et al (2022) similarly engineered the MS and MP nucleotide modifications to their synthesized sgRNA structures and observed increased indel editing for the HBB gene within human primary T cells. By incorporating these alternations within the internal structure of these molecules, they were able to decrease the percentage of off-target indels thus increasing the sgRNA selectively ( Figure 1B ) ( Ryan et al, 2022 ).…”