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Analysis of an apparent mutant/morphant disagreement

Barthelson K, Baer L, Dong Y, Hand M, Pujic Z, Newman M, Goodhill GJ, Richards RI, Pederson SM, Lardelli M. Zebrafish Chromosome 14 Gene Differential Expression in the fmr1 (h u2787) Model of Fragile X Syndrome. Front Genet. 2021 May 31;12:625466. doi: 10.3389/fgene.2021.625466. eCollection 2021.

https://www.frontiersin.org/articles/10.3389/fgene.2021.625466/full

Interesting analysis of an apparent mutant/morphant disagreement finding no genetic compensation experimentally.

"Kok et al. (2015) summarised the puzzling, frequent discordance between phenotypes caused by gene mutations compared to morphant phenotypes caused by reduction of gene expression due to injection of morpholinos. Rossi et al. (2015), then described the phenomenon of “genetic compensation” (now referred to as “transcriptional adaptation” Kontarakis and Stainier, 2020) as contributing to this discordance. As elaborated in a subsequent paper from that laboratory (El-Brolosy et al., 2019), non-sense-medicated decay (NMD) of transcripts with premature termination codons can (in a manner independent of protein feedback loops) increase the abundance of transcripts of genes with homologous sequences that, presumably, are partially functionally redundant and ameliorate the effects of the mutation. The discovery of this phenomenon raises questions regarding the definition of “null” mutant phenotypes and reveals that reducing gene expression using morpholinos may, in some cases, provide more focussed functional effects at the molecular level that are simpler to interpret than those caused by mutations inducing NMD."

"Despite the apparent NMD of fmr1hu2787 transcripts, and the reported milder developmental phenotype of fmr1hu2787 homozygotes relative to individuals in which the function of this gene is suppressed using morpholinos, we did not see evidence for transcriptional adaptation by increased transcription of genes possessing sequences with homology to fmr1, at least at 2 dpf. This illustrates possible variability of the occurrence of the recently discovered transcriptional adaptation mechanism, and that more research is required to understand the factors modulating it."

Generating Zebrafish RNA-Less Mutant Alleles by Deleting Gene Promoters with CRISPR/Cas9

A frameshift mutation did not phenocopy the corresponding Morpholino. To avoid transcriptional adaptation (genetic compensation), the authors made mutants with deleted promoters, eliminating the NMD transcript that would trigger transcriptional adaptation.

Generating Zebrafish RNA-Less Mutant Alleles by Deleting Gene Promoters with CRISPR/Cas9.
Kumari P, Sturgeon M, Bonde G, Cornell RA. Methods Mol Biol. 2022;2403:91-106. doi: 10.1007/978-1-0716-1847-9_8.
https://link.springer.com/protocol/10.1007%2F978-1-0716-1847-9_8

N=1 clinical trials (paper)

Preparing n-of-1 Antisense Oligonucleotide Treatments for Rare Neurological Diseases in Europe: Genetic, Regulatory, and Ethical Perspectives.
Synofzik M, van Roon-Mom WMC, Marckmann G, van Duyvenvoorde HA, Graessner H, Schüle R, Aartsma-Rus A. Nucleic Acid Ther. 2021 Sep 29. doi: 10.1089/nat.2021.0039. Online ahead of print.
https://pubmed.ncbi.nlm.nih.gov/34591693/

Chemistry of Peptide-Oligonucleotide Conjugates: A Review

It appears CPP are becoming a hot topic for reviews. This is a nice open-access example.

Klabenkova K, Fokina A, Stetsenko D. Chemistry of Peptide-Oligonucleotide Conjugates: A Review. Molecules. 2021; 26(17):5420. https://doi.org/10.3390/molecules26175420
https://www.mdpi.com/1420-3049/26/17/5420

Review: Genotype-Phenotype Relationships in the Context of Transcriptional Adaptation and Genetic Robustness

Genotype-Phenotype Relationships in the Context of Transcriptional Adaptation and Genetic Robustness.
Jakutis G, Stainier DYR. Annu Rev Genet. 2021 Jul 27. doi: 10.1146/annurev-genet-071719-020342. Online ahead of print.
https://www.annualreviews.org/doi/pdf/10.1146/annurev-genet-071719-020342

Paper: Induction of cryptic pre-mRNA splice-switching by antisense oligonucleotide

Mostly phosphorothioate work

Ham KA, Keegan NP, McIntosh CS, Aung-Htut MT, Zaw K, Greer K, Fletcher Sue, Wilton SD. Induction of cryptic pre-mRNA splice-switching by antisense oligonucleotides. Sci Rep. 2021;11(1):15137 doi:10.1038/s41598-021-94639-x
https://www.nature.com/articles/s41598-021-94639-x

Genetic compensation in soybean (no Morpholino)

Genetic compensation in soybean: I am putting this in the blog as an example of an organism distant from zebrafish which is also compensating for mutation.

Li X, Fang C, Yang Y, Lv T, Su T, Chen L, Nan H, Li S, Zhao X, Lu S, Dong L, Cheng Q, Tang Y, Xu M, Abe J, Hou X, Weller JL, Kong F, Liu B. Overcoming the genetic compensation response of soybean florigens to improve adaptation and yield at low latitudes. Curr Bio. 2021;[Epub] doi:10.1016/j.cub.2021.06.037
https://www.cell.com/current-biology/fulltext/S0960-9822(21)00832-0

Mutant-morphant mismatch but no genetic compensation

Zebrafish Chromosome 14 Gene Differential Expression in the fmr1 h u2787 Model of Fragile X Syndrome.
Barthelson K, Baer L, Dong Y, Hand M, Pujic Z, Newman M, Goodhill GJ, Richards RI, Pederson SM, Lardelli M. Front Genet. 2021 May 31;12:625466. doi: 10.3389/fgene.2021.625466. eCollection 2021.

https://www.frontiersin.org/articles/10.3389/fgene.2021.625466/full

A study of fmr1 in zebrafish assessed changes in related genes between mutants and wild-types, looking for evidence of genetic compensation. No such changes where found, though the mutant and morphant phenotypes were not in complete concordance.

Morphant vs mutant: "...this knockout line is deemed unsuitable for our study."

Here’s a nice description of why, in this study, morphants are preferable to mutants.

"To determine whether Par-3 is involved in polarizing the dynamics of Notch signaling endosomes, we disrupted the activity of the zebrafish orthologous gene pard3ab (theretofore referred to as par-3) via microinjection of a well-established antisense morpholino oligonucleotide (MO) (19, 20, 41). Despite the fact that a maternal zygotic germline pard3ab knockout has been previously generated, it has grossly normal brain morphology and survives largely to adulthood (42). This phenotype is distinct from defective brain morphology and abnormal proliferation/differentiation states observed in the morphants, suggesting that genetic compensation (43) is at play. Therefore, this knockout line is deemed unsuitable for our study."

Zhao X, Garcia JQ, Tong K, Chen X, Yang B, Li Q, Dai Z, Shi X, Seiple IB, Huang B, Guo S. Polarized endosome dynamics engage cytoplasmic Par-3 that recruits dynein during asymmetric cell division. Sci Adv. 2021;7(24):eabg1244. doi:10.1126/sciadv.abg1244

https://advances.sciencemag.org/content/7/24/eabg1244

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