Abstract
Post-transcriptional modifications on the guide RNAs utilized in the Cas9 system may have the potential to impact the activity of Cas9. In this study, we synthesized a series of tracrRNAs containing N 6-methyadenosine (m6A), a prevalent post-transcriptional modification, at various positions. We evaluated the effect of these modifications on the DNA cleavage activity of Cas9. Our results show that multiple m6As in the anti-repeat region of tracrRNA reduce the DNA cleavage activity of Cas9. This suggests that the m6A-modified tracrRNA can be used for Cas9 only when the number and the position of the modified residue are properly chosen in tracrRNA.
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