Towards targeted engineering of promoters via deletion of repressive cis-regulatory elements

Many plants with enhanced traits have been engineered via the overexpression of target genes conferring specific benefits to farmers and/or consumers. This is most commonly achieved via transgenic overexpression of either a native or a heterologous gene. However, very few of these transgenic crops have a practical impact due to negative public perceptions of conventional genetically modified (GM) crops and financially burdensome regulations. As an alternative approach, scientists have turned to CRISPR-mediated gene editing to generate transgene-free, gain-of-function alleles. In a recently published article in New Phytologist, Wang et al. (2024; doi: 10.1111/nph.20141) demonstrate the potential to generate ideal gain-of-function plants via CRISPR/Cas9-based targeted deletion of repressive cis-regulatory elements (CRE) from native promoters. This study not only provides a generalizable approach towards rationally generating gain-of-function promoter alleles with a solid mechanistic underpinning but also highlights the power of targeting repressive motifs conserved across species to achieve similar phenotypes (increased protein content) in both rice (a monocot) and soybean (a dicot).

‘… this approach is informed by a solid mechanistic understanding of repressive CREs within promoters, presenting a more rational and systematic approach to achieving endogenous gene overexpression.’

There are several ways by which plant scientists have attempted to generate gain-of-function alleles using CRISPR (Fig. 1). The first and most widely adopted approach is unbiased, multiplex, CRISPR-mediated, mutagenesis of a target promoter with multiple guide RNAs (gRNAs) in hope of generating gain-of-function alleles via fortuitous mutagenesis of upstream regulatory sequences. Since the first demonstration of Cas9-driven promoter mutagenesis by Rodríguez-Leal et al. (2017), several groups have utilized this approach to upregulate their genes of interest (Song et al., 2022; Zhou et al., 2023; Karavolias et al., 2024; Patel-Tupper et al., 2024). However, this method tends to result in mostly loss-of-function (knock-out and knock-down) alleles, and some gain-of-function alleles isolated using this method may involve larger-scale rearrangements such as chromosomal inversions (Patel-Tupper et al., 2024). Additionally, the underlying mechanism driving overexpression of the target gene is frequently not understood in this approach, limiting its potential for rational design and engineering of promoters. A second and more recently attempted approach involves the insertion of enhancers identified via genome enrichment or STARR-seq assays followed by subsequent introduction of the enhancer into the target promoter through CRISPR-mediated knock-in. This method is one of the first to demonstrate systematic engineering of promoter overexpression. However, the mechanism by which these enhancers elicit overexpression also remains elusive. In several cases, these enhancers were either found to barely increase expression above endogenous levels for certain genes (Claeys et al., 2024) or result in excessive overexpression that led to stunted growth and sterility (Yao et al., 2024). The third approach involves CRISPR-targeted mutagenesis of upstream open reading frames (uORFs) present in the 5′-UTR to increase translation of the primary ORF (pORF; Zhang et al., 2018). This is a powerful technique for increasing protein expression but cannot be applied to genes lacking uORFs. In the current study by Wang et al., the authors introduce a fourth method to overexpress an endogenous target gene. Unlike most previous methods, this approach is informed by a solid mechanistic understanding of repressive CREs within promoters, presenting a more rational and systematic approach to achieving endogenous gene overexpression (Fig. 1).

Fig. 1

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Achieving overexpression of endogenous target genes via CRISPR-mediated transgene-free approaches. Successful overexpression of endogenous plant genes has been achieved via: (1) unbiased CRISPR-mediated mutagenesis of upstream regulatory sequences; (2) the insertion of known enhancers into gene promoters; (3) the editing of upstream open reading frames (uORFs) in the 5′ UTR; and (4) the deletion of functional repressive motifs in the gene promoter, as demonstrated by Wang et al. in the current study. Nontransgenic rice and soybean with validated phenotypes were generated via systematic identification and targeted deletion of functional repressive motifs. EMSA, electrophoretic mobility shift assay. Created in BioRender: Wang et al. (2024) BioRender.com/s18z317.

Recognizing the importance of improving protein content in major crops, Wang et al. overexpressed NF-YC4, a conserved transcription factor known to enhance leaf and seed protein content (Li et al., 2015), via targeted deletion of repressive RAV1A and WRKY binding motifs from the rice and soybean NF-YC4 gene promoters. Here, the systematic analysis incorporated into the experimental design deserves particular mention: the authors did not blindly assume that all copies of a putative repressor-binding site were functional. While no previous study has provided satisfying conclusions on how to distinguish functional transcription factor binding sites (TFBS) from nonfunctional ones, many studies have highlighted the critical role of DNA shape and flanking sequences in conferring affinity and specificity to transcription factor binding in plants (Zhang et al., 2019; Sielemann et al., 2021; Li et al., 2023). Notably, a study by Zhang et al. (2019) investigating G-box function in soybean promoters has revealed that the presence of a core TFBS motif is not sufficient for function, and that motifs with consequential effects on expression are often bordered by appropriate flanking sequences, which may be highly motif- and context-dependent. Simply deleting all possible copies of putative repressive CREs without first obtaining data about which elements are actually functional may not lead to the desired outcome, resulting in lost time and effort. Wang et al. used a combination of orthogonal luciferase reporter assays and in vitro electrophoretic mobility shift assay (EMSA) experiments, which allowed them to focus on consequential motifs, thereby facilitating the design of suitable gRNAs for CRISPR-based editing in a targeted and efficient manner. It is worth noting here that the authors successfully used a transient luciferase reporter assay in Nicotiana benthamiana leaves to obtain initial readouts of gene expression driven by various NF-YC4 promoter variants. Jores et al. (2021) have previously revealed limitations of studying dicot transcriptional responses in monocots, and vice versa; however, the results of Wang et al. indicate that well-established model systems such as N. benthamiana retain the power to provide accurate readouts on monocot (rice) promoter activity as long as the transcription factors and their cognate TFBS are known to be well conserved across monocots and dicots. The strong correlation between luciferase readouts and leaf transcript levels, as shown for rice NF-YC4 expression, strongly supports this and may spare future researchers the laborious task of isolating protoplasts from their species of interest for initial validation experiments. Thus, researchers aiming to rationally engineer promoters for overexpression may want to consider focusing their efforts on targeting putative repressive sites within promoters that are conserved across plant species. By examining transcript levels as well as protein and carbohydrate content across different tissues, the authors also reveal differences in gene expression enhancement in leaves and seeds, raising important questions of tissue specificity and prompting researchers to consider how one may go about achieving tissue-specific endogenous overexpression of target genes in the future.

Another highlight of the Wang et al. study is the utilization of site-directed nuclease (SDN)-1 type edits to overexpress NF-YC4 in rice and soybean. There are three major types of edits used to categorize CRISPR modification outcomes in plants (Podevin et al., 2013): (1) SDN-1 edits, which include all editing outcomes of double-strand breaks resulting from the plant's native nonhomologous end joining (NHEJ) repair mechanism; (2) SDN-2 edits that utilize a repair template with only a few bases differing from the original sequence; and (3) SDN-3 edits, which involve incorporation of a foreign or exogenous piece of DNA, typically large and considered as GM (Ahmad et al., 2023). Wang et al. generated their NF-YC4 promoter deletions via simultaneous introduction of gRNAs that target promoter segments harbouring functional repressive CREs, resulting in double-strand breaks that were presumably repaired by NHEJ, so the edits are considered SDN-1. After editing occurred in the initially transformed T0 generation, transgene-free plants were recovered in subsequent generations by Mendelian segregation, and these plants are more likely to be regulated as non-GM crops by most countries according to current product-based regulations (Ahmad et al., 2023). Such an approach is not only simple and generalizable but may also accelerate the practical impacts of the research, as these newly generated plants would be exempt from lengthy GM regulatory approval processes. Additionally, targeted deletion of repressive CREs may also minimize unintended pleiotropic effects and help preserve most of the native transcriptional regulation. Although current CRISPR tools may not allow researchers to achieve precise deletion of only the repressive CREs of interest, the results of Wang et al. demonstrate that the deletions need not necessarily be 100% precise for a physiologically relevant and desirable phenotype to be attained. New gene-editing tools are being developed for ever-more precise endogenous DNA sequence manipulation, and this study provides plant scientists with a roadmap for implementing a powerful strategy for targeted promoter engineering and endogenous gene overexpression.