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