Key Points
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Loss-of-function (LOF) technologies are widely used across many model organisms and many fields.
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Although all LOF approaches have the shared goal of perturbing gene function, there are complex differences between approaches that can have a considerable effect on the outcome of experiments.
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The specific properties and effect of each loss-of-function approach depend on the model organism in which they are used.
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Outcomes from LOF experiments depend on the strength and duration of knockdown or knockout and the process targeted (for example, DNA sequence, transcription, mRNA or protein).
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The most appropriate choice of LOF method requires careful consideration and will depend on both the biological question to be answered and the model organism to be used.
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It can be advantageous to apply different LOF approaches in parallel in order to gain greater confidence in results and a deeper understanding of the underlying biology.
Abstract
Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or protein to reduce or to ablate gene function. By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated. Although all LOF methods reduce gene activity, the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes. Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method. The practicality and efficiency of LOF methods also vary considerably between model systems. We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method.
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Acknowledgements
This work was supported by the US National Institutes of Health (NIH) R01HD37047 (G.S.) and 5P01CA120964 (N.P.). Research in D.Y.R.S.' laboratory is funded in part by the DFG, EU and the Max Planck Society. Research in J.Z.'s laboratory is funded by ERC Starting Grant 336860, SFB grant F4710 of the Austrian Science Fund (FWF) and generous institutional support from Boehringer Ingelheim. M.M. is a recipient of a DOC Fellowship of the Austrian Academy of Sciences. S.E.M. is supported in part by NIH NIGMS R01 GM067761 (N.P., PI) and by NCI Cancer Center Support Grant NIH 5 P30 CA06516 (E. Benz, PI). C.A.G. and M.G. are supported in part by NIH DP2OD008586, R01DA036865, R21DA041878, and R21AR065956. N.P. and G.S. are HHMI Investigators. The authors apologize to the authors of the many relevant papers that they were not able to cite owing to length limitations.
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J.Z. is a member of the scientific advisory board of Mirimus Inc., a company that develops RNAi-based reagents and transgenic mice. The other authors declare no competing interests.
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Glossary
- Knockout
-
A genetic perturbation that completely ablates gene function.
- Knockdown
-
A perturbation at the DNA, RNA or protein level that reduces the amount of functional protein.
- Pleiotropic
-
A gene that has roles in more than one stage, tissue or process.
- Off-targets
-
Disruptive effects on gene function at unintended targets.
- Enhancer-trap mutants
-
An exogenous DNA cassette or construct that is used to provide a minimal promoter and reporter such as Gal4 or GFP that, when inserted near a DNA element (such as an enhancer), becomes expressed under the control of that element. It is distinct from a gene trap in that an enhancer trap is not necessarily inserted within a gene.
- Gene-trap mutants
-
An exogenous DNA cassette or construct that is used to provide a reporter such as Gal4 or GFP (for example, as an artificial exon) that, when inserted into a gene (for example, into an intron separating coding exons), becomes expressed under the control of the endogenous promoter of that gene.
- Knock-ins
-
Introduction of a specific exogenous sequence — such as a reporter, selectable marker or engineered mutation — into a specific genomic region, typically a gene region.
- Arrayed format
-
A high-throughput screen format in which each reagent or set of gene-specific reagents (such as a small interfering RNA (siRNA) 'mini-pool') is in a separate well of a micro-well format plate. Positive results of a cell-based assay carried out in an arrayed format can be matched back with reagents (and thus the targeted gene) using a look-up table.
- Pooled format
-
A high-throughput screen format in which gene-specific reagents are introduced into cultured cells en masse and at random, such that the identity of the reagent introduced into any given cell is not known. Positive results of a cell-based assay in a pooled format are typically identified through the sequencing of the starting and final reagent population, for example, reagent sequences extracted from cells at the start of a selection versus sequences remaining following a selection.
- Morphants
-
Organisms that have been treated with a morpholino.
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Housden, B., Muhar, M., Gemberling, M. et al. Loss-of-function genetic tools for animal models: cross-species and cross-platform differences. Nat Rev Genet 18, 24–40 (2017). https://doi.org/10.1038/nrg.2016.118
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DOI: https://doi.org/10.1038/nrg.2016.118
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