Amide Internucleoside Linkages Are Well Tolerated in Protospacer Adjacent Motif-Distal Region of CRISPR RNAs

Abstract

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.

Figure 1.

Structure and sequences of amide-modified crRNAs. The seed region of spacer RNA is underlined, and the repeat region for tracrRNA recognition is shown in outlined font. The position of amide-linked dinucleotide is indicated by bold blue and green fonts.

Figure 2.

EMC assay results normalized to activity of unmodified crRNAs, V0 and H0: (A) crRNAs targeting VEGF-A gene, and (B) crRNAs targeting HPRT1 gene.

Figure 2.

EMC assay results normalized to activity of unmodified crRNAs, V0 and H0: (A) crRNAs targeting VEGF-A gene, and (B) crRNAs targeting HPRT1 gene.

Scheme 1.

Synthesis of amide-linked phosphoramidite dimers for preparation of amide-modified crRNAs.