Pinpoint modification strategy for stabilization of single guide RNA

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 1192; p. 123149
• Main Authors: Takeuchi, Shoko, Yamamoto, Mitsuo, Matsumoto, Satoru, Kenjo, Eriya, Karashima, Masatoshi, Ikeda, Yukihiro
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2022
• Subjects: CRISPR-Associated Protein 9 - chemistry; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems - genetics; CRISPR-Cas9; Mass Spectrometry; Pinpoint modification; RNA Processing, Post-Transcriptional - genetics; RNA Stability - genetics; RNA, Guide, CRISPR-Cas Systems - chemistry; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; Single guide RNA; Stability; MALDI-TOF MS; LC-MS; Stability; Single guide RNA; sgRNA; CRISPR-Cas9; Pinpoint modification; siRNA; Mass spectrometry; HPLC
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2022.123149

•Specific cleavage sites of single guide RNA were identified in biological media.•Combining MALDI- and Q-TOF MS is effective for long length oligonucleotide.•Pinpoint modification allows the stability enhancement of single guide RNA. The clustered regularly interspaced short palindromic repeats-CRISPR associated protein9 (CRISPR-Cas9) system, which includes a single guide RNA (sgRNA) and a Cas9 protein, is an emerging and promising gene editing technology that produces specific changes, including insertions, deletions, or substitutions, in desired targets. This approach can be applied in novel therapeutic areas for multiple cancers and genetic diseases, including Parkinson’s disease, sickle cell disease, and muscular dystrophy. However, there are many limitations to its potential application to therapeutics. CRISPR-Cas9 activity without side effects, delivery of CRISPR-Cas9 to the target cell within the desired tissue including liver, lungs, brain and muscle and the expression of Cas9 endonuclease in the target cell are key factors in achieving therapeutic efficacy. Generally, single-stranded RNA is immediately degraded in cells and biological fluids such as serum, as chemically unmodified single-stranded RNA shows extremely poor stability against nuclease degradation. To overcome this limitation, sgRNA is chemically modified to obtain a highly stable sgRNA for efficient gene editing in cells and in vivo. Here, we identified the cleavage site of sgRNA for pinpoint modification in biological tissues using mass spectrometry and improved stability of pinpoint modified sgRNA in these fluids. Although improved efficiency provided by modified sgRNA has already been reported, we identified the cleavage site by mass spectrometry and revealed that the stability increased with the pinpoint modification strategy for the first time in this study. In future studies, the efficiency of pinpoint modification strategy for the potential application of sgRNA by systematic routes, including intravenous and subcutaneous administration will be assessed.