Innate immune response and off-target mis-splicing are common morpholino-induced side effects in Xenopus George E Gentsch Thomas Spruce Rita S Monteiro Nick DL Owens Stephen R Martin James C Smith 10779/crick.12661445.v1 https://crick.figshare.com/articles/journal_contribution/Innate_immune_response_and_off-target_mis-splicing_are_common_morpholino-induced_side_effects_in_Xenopus/12661445 Antisense morpholino oligomers (MOs) have been indispensable tools for developmental biologists to transiently knock down (KD) genes rather than to knock them out (KO). Here we report on the implications of genetic KO versus MO-mediated KD of the mesoderm-specifying Brachyury paralogs in the frog Xenopus tropicalis. While both KO and KD embryos fail to activate the same core gene regulatory network, resulting in virtually identical morphological defects, embryos injected with control or target MOs also show a systemic GC content-dependent immune response and many off-target splicing defects. Optimization of MO dosage and increasing incubation temperatures can mitigate, but not eliminate, these MO side effects, which are consistent with the high affinity measured between MO and off-target sequence in vitro. We conclude that while MOs can be useful to profile loss-of-function phenotypes at a molecular level, careful attention must be paid to their immunogenic and off-target side effects. 2020-07-17 16:31:14 Brachyury GC content TALEN Xenopus dosage immune response morpholino null mutation off-target splicing Alternative Splicing Animals Embryo, Nonmammalian Female Gene Expression Regulation, Developmental Gene Knockdown Techniques Mesoderm Morpholinos Oligonucleotides, Antisense Xenopus Proteins Xenopus laevis Smith FC001157 SB AS-ack BRF-ack 06 Biological Sciences 11 Medical and Health Sciences Developmental Biology