CRISPR-Cas-mediated unfolded protein response control for enhancing plant stress resistance

Abstract

Plants consistently encounter environmental stresses that negatively affect their growth and development. To mitigate these challenges, plants have developed a range of adaptive strategies, including the unfolded protein response (UPR), which enables them to manage endoplasmic reticulum (ER) stress resulting from various adverse conditions. The CRISPR-Cas system has emerged as a powerful tool for plant biotechnology, with the potential to improve plant tolerance and resistance to biotic and abiotic stresses, as well as enhance crop productivity and quality by targeting specific genes, including those related to the UPR. This review highlights recent advancements in UPR signaling pathways and CRISPR-Cas technology, with a particular focus on the use of CRISPR-Cas in studying plant UPR. We also explore prospective applications of CRISPR-Cas in engineering UPR-related genes for crop improvement. The integration of CRISPR-Cas technology into plant biotechnology holds the promise to revolutionize agriculture by producing crops with enhanced resistance to environmental stresses, increased productivity, and improved quality traits.

Keywords: CRISPR-Cas; crop improvement; endoplasmic reticulum (ER) stress; genome editing; unfolded protein response (UPR).

Figure 1

The UPR in plants: a signaling network coordinating ER homeostasis and stress adaptation. The UPR is activated by the accumulation of unfolded proteins in the ER due to various factors (top left of figure). BiP binds to unfolded proteins, leading to dissociation of IRE1a and IRE1b. Activated IRE1 then cleaves a specific intron from bZIP60u mRNA, generating bZIP60s mRNA. bZIP60s, a potent transcriptional activator, translocates to the nucleus and binds to UPREs and ERSEs in target gene promoters, inducing the expression of stress-responsive genes. In addition to its role in splicing bZIP60 mRNA, activated IRE1 is also involved in a process called Regulated IRE1-dependent decay (RIDD). Under conditions of chronic stress, IRE1 hyper-activates and cleaves additional mRNAs through RIDD. The bZIP17 and bZIP28 pathway is activated by ER stress in plants. Unfolded proteins bind to BiP, causing bZIP17 and bZIP28 to dissociate from the ER membrane. These transcription factors are transported to the Golgi, where proteolytic cleavage mediated by S1P and S2P enzymes releases their N-terminal domains. The N-terminal domains contain the necessary domains for their function as transcription factors. Upon translocation to the nucleus, they bind to ERSE-1 sequences in target gene promoters, inducing the expression of UPR-associated genes encoding ER chaperones and ERAD proteins involved in protein folding, quality control, and degradation within the ER. GCN2 is a kinase activated by dimerization and autophosphorylation in response to endoplasmic reticulum stress. It phosphorylates eIF2α, leading to widespread inhibition of mRNA translation. However, a specific group of uncapped mRNAs with upstream open reading frames (uORFs), such as TBF1 mRNA, are selectively translated. TBF1, a heat-shock factor-like transcription factor, binds to the TL1 cis-element, crucial for inducing BiP2 and CRT3. TBF1 also plays a role in coordinating developmental processes with stress responses, particularly in the growth-to-defense transition. During ER stress, ER chaperones assist in proper protein folding, while ERAD proteins eliminate irreversibly misfolded proteins. ERAD initiation involves OS9 recognizing the N-glycan on a misfolded protein and associating with Sel1L/Hrd3. The Hrd1-Sel1L/Hrd3-OS9 complex, along with UBC32, the E2 enzyme, promotes ubiquitination (Ub) of the misfolded protein for subsequent cytosolic degradation. This process helps restore ER homeostasis by removing unfolded proteins that could disrupt cellular functions.

Figure 2

CRISPR-Cas-Mediated Gene Editing. (A) CRISPR-Cas9-Mediated Gene Editing through Double-Stranded Break (DSB) Repair. The CRISPR-Cas complex cleaves both strands of the target DNA, resulting in a DSB. The repair of the DSB predominantly occurs through two pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ usually restores the original DNA sequence, but it can lead to imperfect repair and DNA insertion or deletion mutations, particularly during intense DSB formation. HR precisely inserts the desired sequence (green sticks) into the genomic region using a DNA donor template with homologous ends to the DSB terminals. (B) DNA base editors for genome editing. Base editing involves a deaminase, usually fused with a Cas9 (nCas9^D10A) nickase, to remove an amino group from a nucleobase on the non-target strand. The deaminated base is then repaired via base excision or nucleotide excision repair, resulting in base transitions or transversions. Depending on the type of deaminase used, base conversion can lead to transitions, such as cytidine deaminase for C/G to A/T in cytosine base editors (CBE), or A/T to G/C in adenine base editors (ABE). Adding uracil DNA N-glycosylase inhibitors (UGI) enhances CBE efficiency. Base transversions can be achieved by adding uracil DNA N-glycosylase (UNG) to CBE (C/G to G/C in CGBE), N-methylpurine N-glycosylase to ABE (A/T to C/G or T/A), or by using UNG alone (G/C to C/G or T/A with GYBE). (C) Prime editing for precise DNA modification. The prime editing utilizes a pegRNA and a reverse transcriptase (RT) enzyme fused to the C-terminal of a nCas9^H840A. It copies genetic information from the 3’ extension of the pegRNA into the nicked end on the non-target strand. By introducing desired genetic changes within the RT template of the 3’ extension, prime editing enables precise genetic modifications at the target site. Prime editing allows for a wide range of precise DNA changes within a genome, including various types of base conversion, DNA insertion, and deletion.

Figure 3

Strategies for prospective editing of ER stress signaling components to enhance stress tolerance. (A) CRISPR-Cas9 can be effectively employed to edit the promoter region of target genes in order to modulate gene expression. The promoter region contains important cis-regulatory elements (CRE, brown and ocher boxes), endoplasmic reticulum stress response elements (ERSEs, blue boxes), and unfolded protein response elements (UPREs, green box). These elements act as enhancers or repressors, playing a crucial role in regulating the transcriptional activity of the gene. By utilizing a multiplex genome editing approach, multiple single-guide RNAs (sgRNAs) can be designed to specifically target distinct ERSEs and UPREs within the promoter region. The CRISPR-Cas9 system, guided by these sgRNAs, induces double-strand breaks at the desired sites in the promoter region, leading to DNA repair mechanisms that can introduce stochastic mutations. These stochastic mutations occurring in the promoter region lead to the generation of alleles with diverse patterns and levels of gene expression. Certain mutations may enhance gene expression, while others may repress it. Implementing this method has the potential to generate a spectrum of phenotypic variations across different lines. (B) CRISPR-Cas9 can be used to manipulate gene translation by targeting upstream open reading frames (uORFs). By utilizing the CRISPR-Cas9 system, specific mutations can be introduced into the start codon region of uORFs, disrupting their inhibitory effects on translation. The translation process of messenger RNA (mRNA) begins when small (light blue) and large (light green) ribosomal subunits scan the mRNA from its 5′ cap (represented by a dark brown circle). The initiation codon, represented by a yellow box, serves as the starting point for translation. However, if the mRNA contains an upstream open reading frame (uORF) represented by a pink rectangle, the ribosome can stall at the uORF. This stalling event leads to the repression of translation of the main open reading frame (mORF) indicated by a blue rectangle. Consequently, the reduced translation of the mORF results in a decreased production of protein products, represented by orange circles. The mutated initiation codon (red rectangle) within uORF regions using the CRISPR-Cas9 inhibits ribosome stalling, resulting in increased production of proteins encoded by the mORF. (C) Strategy to generate truncated UBC32 using CRISPR-Cas9-mediated knockout to enhance BR signaling by stabilizing structurally imperfect, yet biochemically active, bri1 peptides to achieve stress tolerance. The strategy to enhance brassinosteroid (BR) signaling and improve stress tolerance involves the generation of a truncated form of the ubiquitin-conjugating enzyme 32 (UBC32) gene using CRISPR-Cas9-mediated knockout. UBC32 is responsible for encoding an E2 ubiquitin-conjugating enzyme that plays a crucial role in the degradation of the biochemically active but structurally incomplete brassinosteroid insensitive 1 (bri1: bri1-5 or bri1-9) peptide. Through the process of ubiquitination, UBC32 targets the bri1-5 or bri1-9 peptide for 26S proteasome-mediated degradation in the cytosol. However, utilizing the CRISPR-Cas9 to disrupt UBC32 allows for a reduction in the ubiquitination of bri1-5 or bri1-9 peptide, leading to increased stability of the peptide. This enhanced stability contributes to the amplification of BR signaling, thereby improving stress tolerance in plants.