Genome Engineering Technology for Durable Disease Resistance: Recent Progress and Future Outlooks for Sustainable Agriculture

Abstract

Crop production worldwide is under pressure from multiple factors, including reductions in available arable land and sources of water, along with the emergence of new pathogens and development of resistance in pre-existing pathogens. In addition, the ever-growing world population has increased the demand for food, which is predicted to increase by more than 100% by 2050. To meet these needs, different techniques have been deployed to produce new cultivars with novel heritable mutations. Although traditional breeding continues to play a vital role in crop improvement, it typically involves long and laborious artificial planting over multiple generations. Recently, the application of innovative genome engineering techniques, particularly CRISPR-Cas9-based systems, has opened up new avenues that offer the prospects of sustainable farming in the modern agricultural industry. In addition, the emergence of novel editing systems has enabled the development of transgene-free non-genetically modified plants, which represent a suitable option for improving desired traits in a range of crop plants. To date, a number of disease-resistant crops have been produced using gene-editing tools, which can make a significant contribution to overcoming disease-related problems. Not only does this directly minimize yield losses but also reduces the reliance on pesticide application, thereby enhancing crop productivity that can meet the globally increasing demand for food. In this review, we describe recent progress in genome engineering techniques, particularly CRISPR-Cas9 systems, in development of disease-resistant crop plants. In addition, we describe the role of CRISPR-Cas9-mediated genome editing in sustainable agriculture.

Keywords: CRISPR-Cas system; disease resistance; genome editing; pesticide; plant pathogen.

Figure 1

Comparison of different widely used genome editing systems in plants. Site-specific techniques, such as those based on zinc finger nucleases (ZFNs; A) and protein-dependent DNA cleavage systems, such as those based on transcription activator-like effector nucleases (TALENs; B). The two sequence-specific zinc finger proteins flank the target sequence, and FokI nuclease follows the C terminus of each protein. Zinc finger proteins are used to direct FokI dimers to target the specific DNA sites to be cleaved. TALENs consist of two sequence-specific TALEN proteins arranged on the target sequence, with FokI nuclease followed by the C terminus of each protein. The TALEN protein directs FokI dimers to the target specific DNA sites to be cleaved. (C) Illustration of a cluster of the regularly spaced short palindrome repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system. CRISPR-Cas induces double-strand breaks (DSB) in the DNA strand. CRISPR RNA (crRNA) directs the Cas9 protein. Trans-activating CRISPR RNA (tracrRNA) is used to stabilize the structure and activate Cas9 to cut the target DNA. Single guide RNA [sgRNA (Gulabi)] recognizes the target gene, and then the Cas9 protein (green) cuts the two strands of the target DNA at the RuvC and His Asn-His (HNH) domains. The cutting of DNA by Cas9 is dependent on the presence of spacer adjacent motif (PAM) sequences. A 20-base sgRNA defines the binding specificity. In DNA, there are two mechanism of double-strand break (DSB) repair, namely, homology-directed repair (HDR), which is activated in the presence of a template and leads to knock-in or gene replacement, and non-homologous end joining (NHEJ), which is not sufficiently precise and leads to permanent gene knockout. (D) The Cpf1 system. Cpf1 is a two-component RNA programmable DNA nuclease related to CRISPR. The PAM of Cpf1 is TTTN. Cpf1 cuts the target DNA and introduces a DSB, a 5-nt staggered cut at the 5′ end of the T-rich PAM. CRISPR-Cas9 nickase introduces breaks only in the strand complementary to sgRNA. The double nickase with two sgRNAs introduces a staggered DSB into the DNA, after which HDR repairs the DSB. Uracil DNA glycosylase inhibitor (UGI) protein prevents removal of uracil from the DNA and subsequent repair pathways and contributes to increasing the frequency of mutations.

Figure 2

The resistance (R) gene stacking method. (A) Cross-pollinated individuals, based on established trait loci, stack marker-assisted breeding and combine the combined trait loci for marker-assisted selection of the offspring. (B) A single transgene stacking event can be completed by integrating multiple genes into a single stack vector and introducing these simultaneously. (C) Targeted insertion stacking aims to insert new genes near established loci. To stack a large number of genes, this process can be repeated. The stacked genes in B and C are genetically related, such that they can be readily inserted at a single site.

Figure 3

A potential target for applying CRISPR-Cas for engineering of “non-transgenic” disease resistance in plants. In response to pathogen recognition, the plant-induced defense response involves a MAP kinase phosphorylation cascade, which leads to the activation of transcription factors and the expression of defense-related genes. (A) Open reading frame disruption by non-homologous end joining (NHEJ) causes frameshift mutations in susceptibility genes. (B) NHEJ eliminates cis elements to prevent transcription activator-like effector (TALE) activation of susceptibility genes and homology-directed repair (HDR) introduces cis elements, which promotes the TALE-triggered activation of defense genes. (C) Rewriting of transboundary RNAi via HDR-mediated pathogen siRNA targeting. (D) HDR-mediated coding sequence rewriting replaces the effectors produced by pathogens for amino acid residues required for protein recognition, thereby preventing, for example, cleavage or modification. The figures are adopted with modification from those presented by Schenke and Cai (2020).

Figure 4

The mechanisms of base editing. In the absence of double-strand breaks (DSBs), base editing facilitates the introduction of precise point mutations at specified target sites in the genome via nucleotide substitution. (A) Cytidine deaminase base editing (CBE) is performed in conjunction with the use of an APOBEC1 cytidine deaminase, which converts C to U. Subsequently the U-G mismatch is resolved via cellular mismatch repair or base editing mismatch repair machinery that leads to the formation of T-A at the target locus. (B) The adenine base editing (ABE) leads A-T to G-C substitution. After recruiting to the targeted genomic locus, the ABE delaminates targeted A base to I (inosine) leading to I-T base pairing. The cellular mismatch repair mechanism or DNA replication resolves the I-T, forming G-C base pairing.

Figure 5

Prime editing is a recently designed base editing technique that facilitates the accurate substitution, insertion, and deletion of sequences. The fusion of Nickase Cas9 (nCas9) and reverse transcriptase (RT) is the most important element in prime editing. The intended modifications are encoded by the prime editing gRNA, which directs the nCas9-RT complex to the target gene sequence. The DNA is cut by the prime editor, which is then hybridized to the primer-binding site, resulting in reverse transcription. DNA ligation and repair are followed by base pairing of 30 or 50 flaps, resulting in DNA editing.

Figure 6

An overview of genome engineering technology and the potential use of the CRISPR-Cas system in different agriculture science disciplines.