CRISPR/Cas9 has revolutionized gene editing, making it easier than ever to modify DNA with precision. At the heart of this tool is a small but powerful molecule called single guide RNA (sgRNA), which guides the Cas9 enzyme to the exact spot in the genome that needs editing. Ensuring that sgRNA is accurately made and fully understood is essential—especially when it comes to meeting scientific and regulatory standards.

Researchers from GenScript Biotech Corporation have developed an innovative approach to fully sequence 100-nucleotide sgRNAs and larger RNA molecules. Their method uses special enzymes—RNase H and DNAzyme—to cut the RNA at specific sites. This precise cutting makes it possible to analyze the smaller pieces using a technique called liquid chromatography tandem mass spectrometry (LC-MS/MS).

They found that DNAzyme, in particular, was more stable and specific than RNase H, making it ideal for this task. It allowed them to achieve 100% sequence coverage, even identifying small impurities at the beginning of the RNA molecule—something that can affect the accuracy of gene editing.

This method could have major implications for scientists and biotech companies working with CRISPR, providing a reliable way to verify the quality of sgRNAs used in research and therapeutic applications. And because the approach targets a conserved region of the RNA scaffold, it can be used broadly across different sgRNA designs.