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

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.

Figure 1. Overview of loss-of-function approaches

a | Targeting the genome. The modification of the genes themselves can result in loss-of-function (LOF) mutations. These mutations can be induced at random, for example, by using mutagens such as high-energy particles (such as, X-rays and gamma rays), which tend to induce double-strand breaks (DSBs), resulting in some cases in large deletions or complex rearrangements; by using chemical mutagens (such as, ethyl methane sulfonate (EMS) and N-ethyl-N-nitrosourea (ENU)), which tend to result in single base-pair changes; and by using transposons. The possible functional outcomes of classical mutations are many (BOX 1). b | Targeting genes. Zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR–Cas9 and other gene-editing approaches can be used to induce DSBs. When repaired through non-homologous end joining (NHEJ), this can result in small insertions or deletions (indels). Combining one of these targeted approaches to generating DSBs with a homologous donor construct, these methods can be used to induce specific changes (for example, specific single base-pair changes) or deletions (for example, of an entire coding region or functional domain), or can be used to replace genes with marker or other constructs (knock-in). Gene-targeted approaches or transposons can also be used to insert fusion tags such as GFP into specific or random locations, respectively. In addition to (or instead of) disrupting genes, the insertion of tags such as GFP or inteins can be useful for RNA and protein-targeting approaches (parts d and e). c | Targeting transcription. A nuclease-dead version of the Cas9 protein (dCas9) fused to appropriate effector domains can be used in combination with guide RNAs (gRNAs) to target appropriate regions to bring about transcriptional activation (CRISPRa) or transcriptional inhibition (CRISPRi), leading to increased or decreased levels, respectively, of the transcript and hence in most cases, leading to increased or decreased levels of the wild-type protein. d | Targeting RNA. The introduction of RNA interference (RNAi) reagents that target a specific gene (or isoform), or targeting GFP (for GFP-tagged genes) can be used to reduce mRNA levels, leading to a reduction in protein levels. The introduction of morpholinos can lead to a block of translation or splicing of the target mRNA. In either case, some population of mRNA might evade RNAi or morpholino targeting, such that the approaches are likely to result in partial and incomplete LOF. e | Targeting proteins. When available, small-molecule inhibitors can be introduced, resulting in the disruption of protein function, for example, through the occupancy of a substrate-binding site or other disruption of function. Genetically tagging a protein of interest through knock-in can allow for its inducible degradation by recruitment of an effector protein that binds the introduced tag and earmarks the protein for proteolysis. ts, temperature-sensitive; TSS, transcription start site.

Figure 2. Effects of different LOF approaches and potential for compensation

a | Different loss-of-function (LOF) reagents have different effects on RNA and proteins. Null mutations can result in no RNA, RNA of a different length (for example, shorter, as in a small deletion), or a full-length RNA with a nonsense or missense mutation, which may or may not be as stable as the wild-type transcript. In a gene deletion that results in a null mutation, the protein would be absent; however, nonsense or missense mutations can result in the production of a truncated or full-length but non-functional protein, which would also behave genetically as null. Similarly, for hypomorphs, the nature of the allele alone does not tell us the effect on RNA or protein levels, stability, or length. For some hypomorphic alleles, RNA levels are reduced; in others, the protein is wild-type in function but is reduced in abundance, has weak activity compared with wild-type, or has only a subset of the full set of wild-type activities (for example, when one but not all functional domains are disrupted or when a key residue of a protein with both catalytic and structural roles is disrupted). With effective RNA interference (RNAi) reagents, RNA sequences are wild-type but levels are reduced, leading to reduced levels of the protein. With morpholinos, RNA is present but translation or splicing is blocked, leading to reduced levels of the protein. For RNAi and morpholinos, the effectiveness of the reagent (for example, percentage knockdown), as well as the initial abundance and/or the half-life of the protein can affect the protein levels and thus, the timing and severity of a LOF phenotype. With a degron approach, in which proteins are targeted for proteolysis, RNA is present and protein levels are reduced. With a small-molecule approach, protein levels are typically not affected. In this case, some reduced level or subset of wild-type activities could result, for example, from a structural contribution made by protein bound to a small molecule that only affects a catalytic domain, and/or by a population of unbound protein. b | Compensation following acute or long-term LOF disruption. With acute disruption of gene A, the pathway, complex or other activity in which protein A participates is disrupted, leading to the reduction or elimination of the outcome. When cells have time to adapt to a disruption, transcriptional changes such as the upregulation of positively acting factors or the downregulation of negatively acting factors might partially or fully restore the outcome. In addition, cells might accumulate one or more mutations in genes controlling the same activity (for example, activating mutations in positive regulators or inactivating mutations in negative regulators), and/or mutations in alternative pathways or activities that also affect the outcome, leading to partial or full restoration of the outcome.