Poster Presentations
Day 2, June 11（Thu.） Room P (5F 501+502)
- 2P-29
Development of a DNA Cleavage Strategy using CRISPR/Cas9 System for Mass Spectrometry
(AIST)
oYuriko Adachi, Kazumi Saikusa, Megumi Kato
The verification of oligonucleotide sequence in quality control of nucleic acid therapeutics commonly relies on mass spectrometry; however, sequences exceeding 60 nucleotides must be cleaved into shorter fragments before analysis. Although restriction enzymes are conventional DNA cleavage tools for mass spectrometry, precise control over cleavage sites remains difficult. This study describes the development of a CRISPR/Cas9-based DNA cleavage strategy for mass spectrometry, enabling control of DNA fragment lengths. In the CRISPR/Cas9 system, the Cas9 enzyme and a guide RNA form a complex, which can recognize both the protospacer adjacent motif (PAM) and the guide-defined target sequence. Additionally, Cas9 can cleave target DNA lacking a PAM sequence through hybridization with an oligonucleotide containing a PAM sequence (PAMmer). We initially optimized the cleavage conditions for double-stranded DNA containing a PAM sequence using an 80-bp model DNA, achieving CRISPR/Cas9 cleavage efficiency comparable to that of restriction enzymes. We further investigated the characteristics of PAMmers required for CRISPR/Cas9 cleavage of target DNA lacking the PAM sequence. Finally, Cas9-cleaved DNA fragments were detected by mass spectrometry, indicating that this strategy will facilitate characterization of long DNA sequences.