Mara E. Robu, Jon D. Larson, Aidas Nasevicius, Soraya Beiraghi, Charles Brenner, Steven A. Farber, Stephen C. Ekker This study investigates the off-target effects of Morpholino phosphorodiamidate oligonucleotides (MOs) and short interfering RNAs (siRNAs) in zebrafish embryos, focusing on p53 activation as a major mechanism. The authors found that both MOs and siRNAs can induce off-target effects, leading to p53 activation and subsequent cell death. They demonstrated that concurrent knockdown of p53 significantly reduces this off-target cell death. Additionally, they observed the transcription of an N-terminal truncated p53 isoform, which is a diagnostic signature of off-target effects. The study suggests that p53 knockdown could be used to mitigate off-target effects in gene knockdown studies, potentially improving the specificity and reliability of these techniques.Mara E. Robu, Jon D. Larson, Aidas Nasevicius, Soraya Beiraghi, Charles Brenner, Steven A. Farber, Stephen C. Ekker This study investigates the off-target effects of Morpholino phosphorodiamidate oligonucleotides (MOs) and short interfering RNAs (siRNAs) in zebrafish embryos, focusing on p53 activation as a major mechanism. The authors found that both MOs and siRNAs can induce off-target effects, leading to p53 activation and subsequent cell death. They demonstrated that concurrent knockdown of p53 significantly reduces this off-target cell death. Additionally, they observed the transcription of an N-terminal truncated p53 isoform, which is a diagnostic signature of off-target effects. The study suggests that p53 knockdown could be used to mitigate off-target effects in gene knockdown studies, potentially improving the specificity and reliability of these techniques.