Conditional and tissue-specific approaches to dissect essential mechanisms in plant development

Abstract

Reverse genetics approaches are routinely used to investigate gene function. However, mutations, especially in critical genes, can lead to pleiotropic effects as severe as lethality, thus limiting functional studies in specific contexts. Approaches that allow for modifications of genes or gene products in a specific spatial or temporal setting can overcome these limitations. The advent of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technologies has not only revolutionized targeted genome modification in plants but also enabled new possibilities for inducible and tissue-specific manipulation of gene functions at the DNA and RNA levels. In addition, novel approaches for the direct manipulation of target proteins have been introduced in plant systems. Here, we review the current development in tissue-specific and conditional manipulation approaches at the DNA, RNA, and protein levels.

Keywords: CRISPR; CRISPR-Activation; Conditional; Degron; Gene silencing; Nanobodies; Tissue-specific.

Figure 1. Schematic representation of current and future applications of temporally and spatially controlled CRISPR/Cas tools in plants.

(A) Conditional and tissue-specific CRISPR-Cas activity in plants has been obtained via restricted temporal and/or spatial expression of a Cas nuclease. Anti-CRISPR proteins inactivate Cas nuclease activity at the post-translational level. Conditionally destabilized Cas proteins allow for activation and deactivation at the post-translational level. (B) Conventional mutagenesis using WT CRISPR/Cas9 enables gene knockout via DNA double-strand breaks adjacent to the PAM generated by the two Cas9 nuclease domains. Subsequent non-perfect repair leads to indel formation. CRISPR/dead Cas9 (dCas9)-based transcriptional regulation is obtained via fusion of effector domains and allow gene activation or repression. CRISPR/Cas9 nickase (nCas9)-based base editors are composed of a cytidine or adenine deaminase fused to nCas9 and enable the base pair conversion of C·G to T·A or A·T to G·C, respectively. Base editing has not been used tissue-specifically or conditionally in plants yet. CRISPR/Cas13a targets and cleaves single-stranded RNA and allows for knockdown of transcripts which has not been used tissue-specifically or conditionally in plants yet. Applications established tissue-specifically or conditionally in plants in black font; applications not established tissue-specifically or conditionally in plants yet in grey font. GOI: gene of interest; PAM: protospacer adjacent motif.

Figure 2. Schematic representation of targeted protein degradation and delocalization approaches in plants.

(A) temperature-dependent degron approach. The degron cassette consists of an N-terminal sequence encoding a single ubiquitin (Ub), the thermo-labile mouse dihydrofolate reductase (DHFR^ts) containing 16 Lys (K) and destabilizing (R) residues, followed by C-terminal located protein of interest (POI). At the permissive temperature (13 to 16 °C, green) the fusion protein is stable. To make the cassette liable for the proteasomal degradation pathway, it is subjected to co-translational deubiquitylation by deubiquitylating enzymes and Ub-specific processing proteases to reveal the N-degron. The restrictive temperature (27 to 29 °C, red) triggers DHFR^ts flexibility to expose internal K residues, which are recognized by Ub ligases, and targets the POI for the 26S proteasome degradation. (B) TIPI-degron. The N-degron in the fusion protein is covered by green fluorescent protein (GFP). Upon TEV protease cleavage, GFP is removed to expose the N-degron for polyubiquitination and consequent degradation. (C) deGradFP. This system utilizes the SCF complex that consists of endogenous components: the adapter elements for the F-box protein (Skp1), the cullin scaffold (Cul1) and the E2 ligase-recruiting RING protein (Rbx1). The specificity of the substrate is given by the FBP subunit target recognition (F-box) fused to the vhh-GFP4, an anti-GFP nanobody. The GFP-POI is recognized by the F-box/vhh-GFP4, and undergoes polyubiquitination mediated by the E2 ubiquitin protein ligase to finally be degraded in the proteasomal pathway. (D) GFP-Nb. This system relies on the specificity of an anti-GFP nanobody (GFP-Nb) fused to the import signal of the yeast mitochondrial outer membrane protein Tom70p. In the poi complemented mutant background GFP-Nb targets the POI-GFP and re-localizes it to the mitochondria, therefore sequestering the functional POI at an ectopic subcellular localization.