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A prp1 Morpholino injected into zebrafish with mutated prp1 caused significant phenotype at a dose of 6 ng/embryo but had little effect at lower doses (see Supplemental Figure S13). This is a case where the Morpholino-in-mutant experiment not only detected an off-target interaction of the Morpholino but defined a dose where the effect suddenly became strong. Doses of prp1 Morpholino below that threshold produced phenotype at roughly the rate of the standard control oligo injections.

Leighton PLA, Kanyo R, Neil GJ, Pollock NM, Allison WT. Prion gene paralogs are dispensable for early zebrafish development but have non-additive roles in seizure susceptibility. J Biol Chem. 2018 Jun 14. pii: jbc.RA117.001171. doi: 10.1074/jbc.RA117.001171. [Epub ahead of print]
http://www.jbc.org/content/early/2018/06/14/jbc.RA117.001171.abstract

"Consensus guidelines for the use and interpretation of angiogenesis assays" contains a very good discussion of Morpholinos and specificity. Nowak-Sliwinska et al. extensively cite the Stainier et al. "Guidelines for Morpholino Use in Zebrafish". There is a theme in the Morpholino discussion in Nowak-Sliwinska et al.'s paper with which I disagree. They state "However, the best and generally accepted validation for any MO phenotype is confirmation of the same phenotype in a zebrafish genetic mutant." [1] Is this best, or does it exclude a valuable function of Morpholinos? Following Stainier et al., I advocate that performing a specificity control by using a targeting Morpholino in a null background for the same transcript is a better validation for the MO. In a mutant undergoing compensatory changes in gene expression, Morpholinos checked with that method which produce no additional observed effects (in a compensated background) might present with more extreme phenotypes when used in a wild-type background (in the absence of compensation) and reveal useful information about gene function which is obscured in the mutants. Later in their discussion Nowak-Sliwinska et al. do address the utility of morphants whose phenotypes diverge from their corresponding mutants:

"Fourth, recent work has shown that upregulation of related compensating gene family members can sometimes occur in genetic mutants (by mechanisms that are not yet clear), while this does not appear to take place in MOs-injected animals [277], arguably making MOs a better representation of targeted loss of gene function in these cases."

To accept that a Morpholino might be producing accurate transcript-specific information even when mutant and morphant phenotypes differ, a rigorous specificity control is needed. Stainier et al. write: "A decisive approach to determine the optimal sequence and dose of a MO that does not cause off-target effects is to inject the MO into embryos whose genome (and whose mother’s genome, for maternally expressed genes) has been edited so as to eliminate the MO-binding site or to eliminate the function of the target gene" [2]. Using this specificity control allows application of Morpholinos to probe gene functions which are concealed by compensation in some mutants. I argue it is a better validation than observing agreement of mutant and morphant phenotypes because it is applicable to a broader range of genes.

[1] Nowak-Sliwinska P, Alitalo K, Allen E, Anisimov A, Aplin AC, Auerbach R, Augustin HG, Bates DO, van Beijnum JR, Bender RHF, Bergers G, Bikfalvi A, Bischoff J, Böck BC, Brooks PC, Bussolino F, Cakir B, Carmeliet P, Castranova D, Cimpean AM, Cleaver O, Coukos G, Davis GE, De Palma M, Dimberg A, Dings RPM, Djonov V, Dudley AC, Dufton NP, Fendt SM, Ferrara N, Fruttiger M, Fukumura D, Ghesquière B, Gong Y, Griffin RJ, Harris AL, Hughes CCW, Hultgren NW, Iruela-Arispe ML, Irving M, Jain RK, Kalluri R, Kalucka J, Kerbel RS, Kitajewski J, Klaassen I, Kleinmann HK, Koolwijk P, Kuczynski E, Kwak BR, Marien K, Melero-Martin JM, Munn LL, Nicosia RF, Noel A, Nurro J, Olsson AK, Petrova TV, Pietras K, Pili R, Pollard JW, Post MJ, Quax PHA, Rabinovich GA, Raica M, Randi AM, Ribatti D, Ruegg C, Schlingemann RO, Schulte-Merker S, Smith LEH, Song JW, Stacker SA, Stalin J, Stratman AN, Van de Velde M, van Hinsbergh VWM, Vermeulen PB, Waltenberger J, Weinstein BM, Xin H, Yetkin-Arik B, Yla-Herttuala S, Yoder MC, Griffioen AW.
Consensus guidelines for the use and interpretation of angiogenesis assays.
Angiogenesis. 2018 May 15. doi: 10.1007/s10456-018-9613-x. [Epub ahead of print] Review.
https://link.springer.com/article/10.1007/s10456-018-9613-x

[2] 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.
http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.10...

This is an older review (2008), but has a nice discussion of Morpholinos and controls. Photo-Morpholino and Vivo-Morpholino use in zebrafish are not addressed. The newer strategy of using Morpholinos in CRISPR mutants for specificity control is more recent than this paper, as is the understanding of gene compensation in mutants versus relatively uncompensated phenotypes from knockdowns triggered by Morpholino microinjections.

Skromne I, Prince VE.
Current perspectives in zebrafish reverse genetics: moving forward.
Dev Dyn. 2008 Apr;237(4):861-82. doi: 10.1002/dvdy.21484. Review.

https://onlinelibrary.wiley.com/doi/full/10.1002/dvdy.21484

Here is an example of an unexpected splice-modifying outcome. An oligo was targeted to i1e2, which would normally be expected to skip exon 2. Instead, the oligo caused retention of intron 1. This is the usual outcome of an e1i1 oligo, but not of an i1e2 oligo. Figure 5B shows the gel and the RNA map: https://www.cell.com/cms/attachment/2119254818/2091259359/gr5.jpg

Burns DT, Donkervoort S, Müller JS, Knierim E, Bharucha-Goebel D, Faqeih EA, Bell SK, AlFaifi AY, Monies D, Millan F, Retterer K, Dyack S, MacKay S, Morales-Gonzalez S, Giunta M, Munro B, Hudson G, Scavina M, Baker L, Massini TC, Lek M, Hu Y, Ezzo D, AlKuraya FS, Kang PB, Griffin H, Foley AR, Schuelke M, Horvath R, Bönnemann CG. Variants in EXOSC9 Disrupt the RNA Exosome and Result in Cerebellar Atrophy with Spinal Motor Neuronopathy. Am J Hum Genet. 2018 May 3;102(5):858-873. doi: 10.1016/j.ajhg.2018.03.011.

https://www.cell.com/ajhg/fulltext/S0002-9297(18)30100-9

This is an open-access review covering the mechanism of eukaryotic RNA splicing and diseases caused by mutations in regions affecting splicing.

Abramowicz A, Gos M. Splicing mutations in human genetic disorders: examples, detection, and confirmation. J Appl Genet. 2018. doi: 10.1007/s13353-018-0444-7
https://rd.springer.com/article/10.1007%2Fs13353-018-0444-7

Review article
Nan Y, Shang Y-J. Antisense Phosphorodiamidate Morpholino Oligomers as Novel Antiviral Compounds. Front Microbiol. 2018;9:750. doi:10.3389/fmicb.2018.00750
https://www.frontiersin.org/articles/10.3389/fmicb.2018.00750/full

It is always prudent to screen proposed oligo sequence for potential off-target RNA interactions using BLAST. Partial homologies between a Morpholino's inverse complement and an RNA which are reported by BLAST are often in regions of the RNA where binding a Morpholino is unlikely to alter gene expression. If there is significant homology in a region where binding a Morpholino is likely to alter a transcript's expression, have us design another oligo.

Whether a BLAST hit presents a potential off-target effect depends on where the hit is located on an RNA as well as the degree of sequence similarity. Different people have different risk tolerance and you'll have to judge whether the risk is appropriate for your experiment. First BLAST the oligo's target sequence and consider the degree and location of similar sequence prior to ordering an oligo synthesis. Then you will also need to do subsequent empirical testing to check for off-target RNA interactions. Selecting and performing a specificity control experiment is an important step during your Morpholino experiments; specificity controls may include using a second non-overlapping oligo, doing an mRNA rescue, or using the oligo in a mutant that is null for the RNA the Morpholino targets.

BLAST the invert complement of the Morpholino sequence (the oligo's target sequence) in order to find places where the Morpholino might bind to off-target mRNA. For a translation blocker, at least BLAST the transcript sequences. For a splice modifier, at least BLAST genomic sequence. It is possible that a sequence from a 5'-UTR might occur elsewhere near a splice junction, or that a splice junction sequence might occur in a 5'-UTR, so for the most thorough check for unexpected interaction with RNA it is best to BLAST each oligo target against both transcript and genomic sequence.

Remember when searching for homologous targets that Morpholinos will only block translation when targeted to the UTR or first 25 bases of coding sequence. Also, Morpholinos can affect splicing if targeted in introns near intron-exon boundaries. If the Morpholino has homology to an off-target mRNA outside of these limited regions, binding of the oligo to the mRNA is less likely to affect expression of the off-target mRNA (though blocking regulatory sequences [e.g. exonic splice enhancers, 3'-UTR miRNA targets, poly-A signal sequences, etc.] may affect expression).

I usually suggest that the fraction of homologous bases should be below about 80%, but that cutoff % ignores important considerations about the distribution of the mispairs through the oligo. We have long stated that 14 to 15 contiguous bases of homology is the minimum inactivating length for a Morpholino (that is, less than a 14-mer oligo won't efficiently block translation), but if you flank 10 bases of homology with a mispair at either side and then add some runs of homologous bases at the outside borders, you can still get a knockdown. High CG content can make shorter homologous sequences active, since CGs pair with greater stability than ATs; in general, homologous sequences with high predicted Tm should be avoided if they are in an area which might affect gene expression. Recent reports suggest that homologies with as little as 11 bases matched can have off-target effects (see: BLAST homology and specificity controls), though the 11-base sequence reported has an unusually high Tm (see: Paper on Morpholino specificity and CRISPR-MO combination). Five mispairs spread throughout a 25-mer almost always gives loss-of-knockdown (so we sell five-mispair oligos as specificity controls, though there are far better specificity controls). If you place all five mispairs in one end of the oligo, you still get 20 contiguous complementary bases in a 25-mer and those 20 bases would retain considerable antisense activity. My point is that when you find a partially-homologous region, following a rule of thumb like "less than 80% homology is OK" can lead to trouble -- you still need to look at distribution of the mispairs.

Watch for multiple regions of homology, for example 12 bases of homology, 1 mismatch, and 12 more bases of homology. If you are just looking for contiguous homology you would interpret this as 12 contiguous bases of homology, but it is really a single-mismatch 25-base target that would very likely lead to strong knockdown by the Morpholino if it is in a place where the bound Morpholino would alter gene expression.

Here are a few more factors to consider: losing a C-G pair impacts the oligo activity more than losing an A-T pair (three H-bonds for G-C compared to two for A-T) and watch for forming G-T pairs, which are non-Watson-Crick pairing but still form two hydrogen bonds.

The BLAST algorithm is not an exhaustive sequence search, so it is not surprising you see some differences between different BLASTs of the same sequence. Screening using several databases is best and even with the same genome build, they will often produce different results; it really depends how much of your time you make available for the screen. BLASTING a sequence can sometimes catch a problem, but it is an endless rabbit-hole to explore so you have to set a limit on how long you will search a given sequence. In the end, a good physical specificity control should tell you if the targeting oligo is working specifically. BLASTing just helps eliminate some oligos up-front that will not survive the specificity control challenge.

"Due to genetic compensation, phenotypically wild-type mutants can become refractive to morpholino-induced phenotypes, providing a critical test both for genetic compensation and for the specificity of morpholino phenotypes."

Balciunas D. Fish mutant, where is thy phenotype? PLoS Genet. 2018;14(2):e1007197.
https://doi.org/10.1371/journal.pgen.1007197

This is an interesting technique for later-stage zebrafish embryo treatment, involving pulsed injection of a mixture of Morpholino and Endo-Porter into the heart. I think this might push the limit of solubility for many Morpholino sequences. They are reporting good systemic delivery -- see the video for fluorescent images of the oligo distribution.

Konantz J, Antos CL. Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva. J Vis Exp. 2014;88:e51595 doi:10.3791/51595
https://www.jove.com/video/51595/reverse-genetic-morpholino-approach-usi...

This one looks interesting. Morpholino and 2'-MOE activities are compared in SMA models.

United States Patent Application 20180030443
February 1, 2018
Inventors: Burghes; Arthur; (Columbus, OH) ; Porensky; Paul; (Worthington, OH) ; Kaspar; Brian; (New Albany, OH)

NON-IONIC, LOW OSMOLAR CONTRAST AGENTS FOR DELIVERY OF ANTISENSE OLIGONUCLEOTIDES AND TREATMENT OF DISEASE