Transcriptome-wide RNA off-target effect of FNLS-YE1

Recent studies reported that cytosine base editors could induce Cas9-independent DNA off-target effects, as well as transcriptome-wide RNA off-target effects. However, some improved base editors, including FNLS-YE1, induced no detectable off-target effects in mouse embryos and human cell lines. Here we further investigated whether FNLS-YE1 induces off-target effect in human embryos. For Cas9-independent RNA off-target effect, we performed single-cell RNA sequencing on the GFP-only, FNLS-YE1+PCSK9 sgRNA+GFP, and FNLS-BE3+PCSK9 sgRNA+GFP -injected blastomeres, together with corresponding un-injected blastomeres two days after injection. We found the background level of RNA SNVs and no canonical C-to-U pattern in the FNLS-YE1 injected blastomeres compared with GFP injected blastomeres. By contrast, FNLS-BE3 induced severe RNA off-target effects and canonical C-to-U pattern in human blastomeres, consistent with previous studies. Taken together, these results demonstrated that FNLS-YE1 induces no RNA off-target effect in early human embryos.