Loss-of-function genetic tools for animal models: cross-species and cross-platform differences

Housden, Benjamin E.; Muhar, Matthias; Gemberling, Matthew; Gersbach, Charles A.; Stainier, Didier Y. R.; Seydoux, Géraldine; Mohr, Stephanie E.; Zuber, Johannes; Perrimon, Norbert

doi:10.1038/nrg.2016.118

Cited by 175 publications

(154 citation statements)

References 239 publications

(235 reference statements)

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“…The ability to specifically reduce the expression of a particular coding or noncoding gene is fundamental for establishing its loss-of-function phenotype in cells and organisms, and is frequently the only way to infer its function. Three popular strategies for doing this are RNA interference (RNAi), locked nucleic acid (LNA) hybridization and the CRISPR/Cas9 system (1). RNAi uses small interfering 5 RNA oligonucleotides to deplete target transcripts by triggering their degradation (2).…”

Section: Introductionmentioning

confidence: 99%

Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis

Stojic

Lun

Mangei

et al. 2017

Preprint

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Loss-of-function (LOF) methods, such as RNA interference (RNAi), antisense oligonucleotides or CRISPR-based genome editing, provide unparalleled power for studying the biological function of genes of interest. When coupled with transcriptomic analyses, LOF methods allow researchers to dissect networks of transcriptional regulation. However, a major concern is nonspecific targeting, which involves depletion of transcripts other than those intended. The off-target effects of each of these common LOF methods have yet to be compared at the whole-transcriptome level. Here, we systematically and experimentally compared non-specific activity of RNAi, antisense oligonucleotides and CRISPR interference (CRISPRi). All three methods yielded non-negligible offtarget effects in gene expression, with CRISPRi exhibiting clonal variation in the transcriptional profile. As an illustrative example, we evaluated the performance of each method for deciphering the role of a long noncoding RNA (lncRNA) with unknown function. Although all LOF methods reduced expression of the candidate lncRNA, each method yielded different sets of differentially expressed genes upon knockdown as well as a different cellular phenotype. Therefore, to definitively confirm the functional role of a transcriptional regulator, we recommend the simultaneous use of at least two different LOF methods and the inclusion of multiple, specifically designed negative controls.

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Section: Introductionmentioning

confidence: 99%

Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis

Stojic

Lun

Mangei

et al. 2017

Preprint

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“…Molecular profiling and modeling have made important contributions to understanding how genes involved in immunity are influenced. For example, RNA interference (RNAi), morpholinos, chemical inhibitors and hypomorphic mutations often lead to partial suppression of gene function, whereas null mutations can ablate gene function (Housden et al, ). We also know that a host of other factors such as age (Zhang, Drake, Morrison, Oberley, & Kregel, ), metabolism (Boei et al, ), prior exposure (Louis, Bhagooli, Kenkel, Baker, & Dyall, ), and environmental condition (Mangino, Roederer, Beddall, Nestle, & Spector, ) can influence these processes.…”

Section: Discussionmentioning

confidence: 99%

Complex immune responses and molecular reactions to pathogens and disease in a desert reptile (Gopherus agassizii)

Drake

Aiello

Bowen

et al. 2019

Ecology and Evolution

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Immune function plays an important role in an animal's defense against infectious disease. In reptiles, immune responses may be complex and counterintuitive, and diagnostic tools used to identify infection, such as induced antibody responses are limited. Recent studies using gene transcription profiling in tortoises have proven useful in identifying immune responses to various intrinsic and extrinsic stressors. As part of a larger experiment with Mojave desert tortoises ( Gopherus agassizii ), we facilitated the transmission of the pathogenic bacteria, Mycoplasma agassizii (Myag), to naïve adults and measured innate and induced immune reactions over time. Specifically, we evaluated clinical condition, presence of Myag in the nasal/oral cavity, induced antibody responses specific to Myag, and measured molecular reactions (gene transcript profiles) in 15 captive tortoises classified as naïve, exposed, or infected and 14 wild tortoises for comparison. Myag was confirmed inside the nasal/oral cavity in exposed tortoises within 30–60 days of introduction to infected animals, yet we did not detect Myag specific induced antibody responses in these individuals until 420–595 days post exposure. Surprisingly, we found no overall differences in the gene transcript profiles between our experimental treatment groups throughout this study. This work highlights the complexities in assessing immune function and diagnosing pathogen related infections in tortoises and other reptiles.

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“…"knockout", which aims to inactivate the gene of interest by introducing mutations using techniques such as CRISPR/Cas9, and "knockdown", which aims to abrogate the expression of the gene of interest by employing methods such as siRNA, CRISPRi or Morpholino oligomers (for a recent review, see (Housden et al, 2017)).…”

Section: Introductionmentioning

confidence: 99%

Deletion of MOrpholino Binding Sites (DeMOBS) to Assess Specificity of Morphant Phenotypes

Cunningham

Bellipanni

Habas

et al. 2020

Preprint

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Two complimentary approaches are widely used to study gene function in zebrafish:induction of genetic mutations, usually using targeted nucleases such as CRISPR/Cas9, and suppression of gene expression, typically using Morpholino oligomers. Neither method is perfect. Morpholinos (MOs) sometimes produce offtarget or toxicity-related effects that can be mistaken for true phenotypes.Conversely, genetic mutants can be subject to compensation, or may fail to yield a null phenotype due to leakiness. When discrepancy between mutant and morpholino-induced (morphant) phenotypes is observed, experimental validation of such phenotypes becomes very labor intensive. We have developed a simple genetic method to differentiate between genuine morphant phenotypes and those produced due to off-target effects. We speculated that indels within 5' untranslated regions would be unlikely to have a significant negative effect on gene expression.Mutations induced within a MO target site would result in a Morpholino-refractive allele thus suppressing true MO phenotypes whilst non-specific phenotypes would remain. We tested this hypothesis on one gene with an exclusively zygotic function, tbx5a, and one gene with strong maternal effect, ctnnb2. We found that indels within the Morpholino binding site are indeed able to suppress both zygotic and maternal morphant phenotypes. We also observed that the ability of such indels to suppress Morpholino phenotypes does depend on the size and the location of the deletion.Nonetheless, mutating the morpholino binding sites in both maternal and zygotic genes can ascertain the specificity of morphant phenotypes.

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Loss-of-function genetic tools for animal models: cross-species and cross-platform differences

Cited by 175 publications

References 239 publications

Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis

Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis

Complex immune responses and molecular reactions to pathogens and disease in a desert reptile (Gopherus agassizii)

Deletion of MOrpholino Binding Sites (DeMOBS) to Assess Specificity of Morphant Phenotypes

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