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Oocyte microinjection in situ (OMIS): Morpholinos in zebrafish oocytes in the ovary

Wu X, Shen W, Zhang B, Meng A. The genetic program of oocytes can be modified in vivo in the zebrafish ovary. J Mol Cell Biol. 2018 Jul 28. doi: 10.1093/jmcb/mjy044. [Epub ahead of print]

https://academic.oup.com/jmcb/article-lookup/doi/10.1093/jmcb/mjy044

"Furthermore, maternal knockdown of dnmt1 by antisense morpholino via OMIS results in a dramatic decrease of global DNA methylation level at the dome stage and causes embryonic lethality prior to segmentation period."

Targeting non-coding RNAs: strategies and what we need to design an oligo

Morpholinos have been used to alter activity of non-coding RNAs. A citation list is here:
"Other" targets: ncRNA, repeated elements, etc.

Targeting non-coding RNAs presents special problems. Ideally, we need to know where on the non-coding RNA an activity is located that we can block. You will need to tell us where you want the oligo targeted and we’ll try to find the best oligo sequence targeting that location.

For targeting miRNAs, we target to alter the stem-loop structure of the pri-miRNA so that it will not be cleaved by the double-strand nucleases needed for maturing the miRNA (e.g. Drosha, Dicer). Our Design Request Website has a selection available for designing Morpholinos targeting miRNA (https://oligodesign.gene-tools.com/request/).

If the non-coding RNA undergoes splicing, then we can potentially modify the splicing with a Morpholino to excise an exon or insert an intron. In some cases this has been used productively to alter the activity of a non-coding RNA. Targeting a Morpholino to splicing of a non-coding RNA follows the same rules as for targeting a pre-mRNA; typically we target mostly-intronic sequence at a splice junction. Our Design Request Website has a selection available for designing Morpholinos targeting splicing.

We can also directly target activities of non-coding RNAs involving complementary sequence interactions, secondary structure or “sponge” activity. For instance, perhaps the non-coding RNA has a sequence motif that is complementary to another RNA and there is an activity caused by the interaction of the non-coding RNA and the other RNA by complementary base pairing; in that case, we can target a Morpholino oligo across that complementary motif the block the interaction of the two RNAs. Perhaps instead the non-coding RNA's activity is due to a region of secondary structure, such as a crucial stem-loop; in that case, we can target a Morpholino to the stem in order to invade the stem structure, displacing one strand of the stem from the other strand and altering the secondary structure and hopefully altering the activity of that region of the non-coding RNA. Perhaps the non-coding RNA is binding a particular protein or miRNA, acting as a "sponge"; covering the binding site of the protein or miRNA with a Morpholino might productively block the interaction of the RNA with its binding partner. Our Design Request Website has a selection available for designing Morpholinos “Other”; this is an appropriate place to submit sequence for non-coding RNA targets.

To block any of these activities with a Morpholino oligo, we need to know where the activity is located on the non-coding RNA. Recall that Morpholinos do not degrade their RNA targets, so randomly targeting a Morpholino to a non-coding RNA is unlikely to alter the activity of the non-coding RNA. The chances are good that a random target will be distant from the site of the non-coding RNA's activity. So, to design a useful Morpholino for a non-coding RNA we need to know where that activity is located in the non-coding RNA sequence (the base-pairing region, the stem-loop, the binding site, etc.). Please send some sequence for the non-coding RNA with the RNA sequence in UPPER CASE and the active region in lower case, like this:

ATCGTCTATTGTTTCAACTTTTTcgtgctttgatacgcgcgtgatGCTAATACGATTTACTACCATATGAG

We'll look for a good oligo sequence complementary to most or all of the lower case region.

Watch gene expression start to happen in an embryo

Watching dynamics of RNA expression - paper describing a visualization technique.

Movie 1: https://www.biorxiv.org/highwire/filestream/112058/field_highwire_adjunct_files/3/366468-4.mp4

Movie 1 from supplemental information: in this zebrafish embryo, DNA is stained with red fluorescence. Carboxyfluoresceinaed Morpholino oligos targeting dre-miR-430 emit visible green fluorescence when they reach sufficient localized concentration. In red you can watch condensation of chromosomes, mitosis, and loosening of the chromatin. After a few divisions you will see green dots appear where groups of miR430 genes are being transcribed and capturing fluorescent-labeled Morpholinos. Each nucleus contains two dots, the maternal and paternal chromosomes revealing the site of miR430 transcription. The green dots disappear as the red chromosomes condense out of the chromatin for mitosis and gene expression halts for division. This movie shows the early-to-mid blastula stages and the onset of zygotic transcription occurs at mid-blastula, so you don't see the green dots appear during the first few cell divisions; early on the cells are expressing maternal mRNAs that are already present in the egg. The onset of zygotic transcription is where the embryo begins to rely on its own genome.

The paper: https://www.biorxiv.org/content/early/2018/07/15/366468

Hadzhiev Y, Qureshi H, Wheatley L, Cooper L, Jasiulewicz A, Nguyen HV, Wragg J, Poovathumkadavil D, Conic S, Bajan S, Sik A, Hutvagner G, Tora L, Gambus A, Fossey JS, Mueller F. A cell cycle-coordinated nuclear compartment for Polymerase II transcription encompasses the earliest gene expression before global genome activation. BioRXive. 2018;[Epub] doi:doi.org/10.1101/366468.

Morpholino duration of effect

A researcher asked about the half-life of the Morpholino. This is much of my response.

The half-life of the molecule is not very useful for experiment planning. The oligos do not degrade (Hudziak RM et al. 1996, Youngblood DS et al. 2007), but their activity is temporarily lost when they bind to complementary RNA; that binding rate is what sets the trajectory of biological activity. Eventually the RNA will degrade off the oligo and release it, but the RNA footprint can be protected from nuclease activity by the Morpholino so this is a slow process, leading to a persistent background of activity well below experimental utility (but reported as weak splice-modifying activity over three months after a single dose in mice, Wells 2008). We typically see about four days of useful knockdown in cultured cells or systemically with a Vivo-Morpholino. Larger doses of Morpholino will persist longer, but the resulting higher oligo concentration in cells might lead to some off-target RNA interaction (and the dose of a Vivo-Morpholino will be limited by toxicity). The rate of new transcription of a particular RNA is an important factor for the duration of a Morpholino knockdown; slow transcription helps the oligo activity persist longer, while rapid transcription can swamp the oligo quickly, leading to a short knockdown. The turnover rate of the protein affects how soon you can detect the knockdown; the oligo might halt transcription, but the protein concentration decreases as a function of its degradation and in some cases can be fairly slow.

Hudziak RM, Barofsky E, Barofsky DF, Weller DL, Huang SB, Weller DD. Resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation. Antisense Nucleic Acid Drug Dev 1996 Winter;6(4):267-72.

Youngblood DS, Hatlevig SA, Hassinger JN, Iversen PL, Moulton HM. Stability of cell-penetrating Peptide-morpholino oligomer conjugates in human serum and in cells. Bioconjug Chem. 2007 Jan-Feb;18(1):50-60.
(note the persistence of the signals corresponding to the mass of the bare oligo without peptide)

Wells DJ. Gene doping: the hype and the reality. Br J Pharmacol. 2008 Jun;154(3):623-31. Epub 2008 Apr 21.

Off-target Morpholino interaction reported based on mutant and 6ng dose

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" with MO discussion

"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...

Review (2008): Current perspectives in zebrafish reverse genetics

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

An i1e2 Morpholino triggering intron 1 retention: unusual splice outcome

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

Splicing mutations in human genetic disorders: Review

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

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