Engineering drought and salinity tolerance traits in crops through CRISPR-mediated genome editing: Targets, tools, challenges, and perspectives

Abstract

Prolonged periods of drought triggered by climate change hamper plant growth and cause substantial agricultural yield losses every year. In addition to drought, salinity is one of the major abiotic stresses that severely affect crop health and agricultural production. Plant responses to drought and salinity involve multiple processes that operate in a spatiotemporal manner, such as stress sensing, perception, epigenetic modifications, transcription, post-transcriptional processing, translation, and post-translational changes. Consequently, drought and salinity stress tolerance are polygenic traits influenced by genome-environment interactions. One of the ideal solutions to these challenges is the development of high-yielding crop varieties with enhanced stress tolerance, together with improved agricultural practices. Recently, genome-editing technologies, especially clustered regularly interspaced short palindromic repeats (CRISPR) tools, have been effectively applied to elucidate how plants deal with drought and saline environments. In this work, we aim to portray that the combined use of CRISPR-based genome engineering tools and modern genomic-assisted breeding approaches are gaining momentum in identifying genetic determinants of complex traits for crop improvement. This review provides a synopsis of plant responses to drought and salinity stresses at the morphological, physiological, and molecular levels. We also highlight recent advances in CRISPR-based tools and their use in understanding the multi-level nature of plant adaptations to drought and salinity stress. Integrating CRISPR tools with modern breeding approaches is ideal for identifying genetic factors that regulate plant stress-response pathways and for the introgression of beneficial traits to develop stress-resilient crops.

Keywords: CRISPR/Cas; drought tolerance; genome editing; polygenic traits; salt tolerance; trait introgression.

Figure 1

Plant responses to drought stress. (A–E) Four critical processes involved in plant drought-stress tolerance after sensing the stress (A) are depicted, along with major changes used by plants in each of the processes, which include avoidance (B), tolerance (C), escape (D), and recovery (E). Specific plant species may respond to drought stress differently, and plants use more than one type of process for growth and survival.

Figure 2

Overview of salinity stress sensing, signaling, and response in plants. Salinity stress is mainly caused by toxic sodium (Na⁺), potassium (K⁺), and chloride (Cl⁻) ions from sodium chloride (NaCl) and potassium chloride (KCl). In the current model, plant sensing, signaling, and response in the context of Na⁺ and K⁺ cytotoxicity are illustrated. Descriptions or full forms of the TF, gene, and family abbreviations are provided in the main text and supplemental information.

Figure 3

Basics of the CRISPR/Cas9 system, Cas variants, and orthologs. (A) Mode of action of the CRISPR/Cas9 system is explained using SpCas9 as a model. The domain organization of the Cas9 enzyme consists of the recognition lobe (REC1, REC2, REC3), bridge helix (Arg), two nuclease domains (HNH, RuvC), and C-terminal protospacer adjacent motif (PAM) interacting domain (CTD). A ribonucleoprotein complex of the Cas9 enzyme (fully functional or impaired form, nickase or dead) with a single guide RNA (sgRNA) searches for the complementary target DNA region. Recognition of target DNA containing the PAM leads to structural rearrangements in Cas9, forming an R-loop in the Cas9-sgRNA-Target DNA complex. Two functional nuclease domains of wild-type Cas9 generate double-stranded breaks (DSBs) in the target DNA. The error-prone repair of DSBs produces insertions or deletions (indels), causing knockout of the encoded genes. (B) Cas variants developed for CRISPR-based tool development. (C) Cas orthologs used for CRISPR-based tool development. N is any nucleotide, R is A/G, M is A/C, D is A/G/T, B is G/T/C, W is A/T, V is G/C/A, R is A/G, and Y is C/T.

Figure 4

CRISPR-based tools and their applications for plant GE. CRISPR-based tools and their applications for DNA modification (indel generation), precise nucleotide substitution, random mutagenesis of a targeted region for novel allele generation, regulation of gene expression, RNA targeting, and CRISPR-based mutant library generation are summarized. Relevant information and references for Cas variants and orthologs are provided in the supplemental information.

Figure 5

Overview of the integration of genomic-assisted breeding and other approaches with CRISPR technology to develop drought/salinity-tolerant crops. Known positive and negative regulators of drought (blue box) and salinity (yellow box) stress tolerance are ideal targets for crop improvement. Descriptions of different aspects depicted here are provided in the main text.

Figure 6

Strategies proposed for the engineering of plant tolerance to drought and salinity stress using CRISPR-based tools. (A) Single or multiple genetic elements can be targeted using desired CRISPR tools. Genes from different pathways responsible for drought or salinity tolerance in plants can be either knocked out (negative element: target 1 or 5, red arrow) or knocked in (positive element: target 2, green arrow). In hypothetical pathway 2, the thickness of the green arrows depicts the major or minor role of the edited target gene in the stress condition. Epigenetic modifiers and CRISPR activation/interference (CRISPRa/CRISPRi) tools provide another means of regulating gene expression without introducing any mutations. In addition, base substitution tools such as base editors enable editing of cis-regulatory elements or generation of novel alleles. Targeting a single gene from a specific pathway may not be sufficient for developing stress tolerance in the case of polygenic traits like drought and salinity. Therefore, targeting of multiple genes from functionally related pathways would be ideal for achieving improved drought and salinity stress tolerance. (B) Differentially expressed genes found in comparative omics analyses of stress-tolerant and sensitive cultivars are ideal candidates for GE to enable further characterization and exploitation in modern breeding. Details are provided in the supplemental information. (C) Detoxification strategies are described in the main text. (D) In the salt gland model, HKT1 transports Na⁺ and NRT transports Cl⁻ to the gland cell; i.e., the epidermal bladder cell. NHX1 and CLC sequester excess Na⁺ and Cl⁻ in the vacuole. H-ATPase and V-ATPase generate proton gradients to drive ion transport.