I have had many discussions about specificity with a Morpholino user who does very careful BLAST analysis of each oligo prior to ordering. I had previously suggested that about 14 bases of complementary (without significant flanking complementarity) is probably an acceptable level of stability for off-target interactions found by BLAST. However, in light of a new report I agreed that a 13-base complementarity at a splice junction is enough to raise concern. Here is an excerpt of my response to her.
My suggestion that you ignore matches of less than 14 contiguous bases needs revision based on a new paper (1). They claim effects down to 11 bp match. The methods and analysis in the paper look good to me, and so for now I must accept their conclusion that, for their particular targeting sequences, the 11 bp match caused off-target effects.
However, we need to make a distinction between BLAST screening and actual specificity controls. The BLAST improves the chances that the oligo will pass muster when doing specificity controls, but it is the controls that (we hope and strive to) reveal the actual biological effect of an oligo. The BLAST is a good guide, but too much emphasis there can prevent getting on with the real experiment. That said, I think you are doing this well and likely saving money on synthesis of some oligos that would present problems later. A prudent level of pre-synthesis screening probably lies somewhere between accepting 14 and 11 base pairs.
Here is my brief blog post on the paper that has made me suggest being more stringent at the BLAST stage, with a relevant quote from the end of the paper:
Paper on Morpholino specificity and CRISPR-MO combination
Regarding controls, I now favor testing a Morpholino in a null mutant for the oligo's target RNA. In the genetically-compensated background of a null mutant, a Morpholino specific for its RNA target should have no effect and that is good evidence for its specificity. Subsequent testing of the Morpholino in a wild-type (uncompensated) organism reveals the outcome of acute change in gene expression without being obscured by compensation (2). A potential difficulty is that the mutant must be a true null (3). The two-nonoverlapping olgo experiment is my second choice and provides enough information in most circumstances, but it is still possible for several oligos to have the same phenotypic outcome though some or all of that outcome is due to interaction with a off-target RNA (4). RNA rescues are strong, giving good evidence for specificity when they work, but they cannot always work due to confounding effects of ectopic expression (in terms of time or place) of the rescue RNA. I stand by my position that the five-mispair experiment has little value.
Additional note 8 Feb 2018: This zebrafish community paper (5) is relevant to anyone working with Morpholinos and running specificity controls.
(1) Joris M, Schloesser M, Baurain D, Hanikenne M, Muller M, Motte P. Number of inadvertent RNA targets for morpholino knockdown in Danio rerio is largely underestimated: evidence from the study of Ser/Arg-rich splicing factors. Nucleic Acids Res. 2017;[Epub ahead of print] doi:10.1093/nar/gkx638.
(2) Rossi A, Kontarakis Z, Gerri C, Nolte H, Hölper S, Krüger M, Stainier DYR. Genetic compensation induced by deleterious mutations but not gene knockdowns. Nature. 2015 Aug 13;524(7564):230-3. doi: 10.1038/nature14580. Epub 2015 Jul 13.
(3)Novodvorsky P, Watson O, Gray C, Wilkinson RN, Reeve S, Smythe C, Beniston R, Plant K, Maguire R, M K Rothman A, Elworthy S, van Eeden FJ, Chico TJ. klf2ash317 Mutant Zebrafish Do Not Recapitulate Morpholino-Induced Vascular and Haematopoietic Phenotypes. PLoS One. 2015 Oct 27;10(10):e0141611. doi: 10.1371/journal.pone.0141611. eCollection 2015.
"In summary, our work shows that even in the face of clear evidence of a potentially disruptive mutation induced in a gene of interest, it is currently very difficult to be certain that this leads to loss-of-function, and hence to be confident about the role of the gene in embryonic development."
(4) Coffman JA, Dickey-Sims C, Haug JS, McCarthy JJ, Robertson AJ. Evaluation of developmental phenotypes produced by morpholino antisense targeting of a sea urchin Runx gene. BMC Biol. 2004 May 7;2:6.
(5) Stainier DYR, Raz E, Lawson ND, Ekker SC, Burdine RD, Eisen JS, Ingham PW, Schulte-Merker S, Yelon D, Weinstein BM, Mullins MC, Wilson SW, Ramakrishnan L, Amacher SL, Neuhauss SCF, Meng A, Mochizuki N, Panula P, Moens CB. Guidelines for morpholino use in zebrafish. PLoS Genet. 2017 Oct 19;13(10):e1007000. doi: 10.1371/journal.pgen.1007000. eCollection 2017 Oct.
Earlier blog post on using a Morpholino in a mutant (from a conversation with Martin Blum): Validating Morpholino phenotypes with CRISPRs
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