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To meet increasing global food demand, breeders and scientists aim to improve the yield and quality of major food crops. Plant diseases threaten food security and are expected to increase because of climate change. CRISPR genome-editing technology opens new opportunities to engineer disease resistance traits. With precise genome engineering and transgene-free applications, CRISPR is expected to resolve the major challenges to crop improvement. Here, we discuss the latest developments in CRISPR technologies for engineering resistance to viruses, bacteria, fungi, and pests. We conclude by highlighting current concerns and gaps in technology, as well as outstanding questions for future research.

Cotton leaf curl disease (CLCuD) is one of the most economically important diseases and is a constraint to cotton production in major producers, Pakistan and India. CLCuD is caused by monopartite plant viruses belonging to the family Geminiviridae (genus Begomovirus), in association with an essential, disease-specific satellite, Cotton leaf curl Multan betasatellite (CLCuMuB) belonging to a newly-established family Tolecusatellitidae (genus Betasatellite). CLCuMuB has a small genome (ca. 1350 nt) with a satellite conserved region, an adenine-rich region and a single gene that encodes for a multifunctional βC1 protein. CLCuMuB βC1 protein has a major role in pathogenicity and symptom determination, and alters several host cellular functions like autophagy, ubiquitination, and suppression of gene silencing, to assist CLCuD infectivity. Efficient trans-replication ability of CLCuMuB with several monopartite and bipartite begomoviruses, is also associated with the rapid evolution and spread of CLCuMuB. In this article we comprehensively reviewed the role of CLCuMuB in CLCuD, focusing on the βC1 functions and its interactions with host proteins.

Background Geminiviruses cause damaging diseases in several important crop species. However, limited progress has been made in developing crop varieties resistant to these highly diverse DNA viruses. Recently, the bacterial CRISPR/Cas9 system has been transferred to plants to target and confer immunity to geminiviruses. In this study, we use CRISPR-Cas9 interference in the staple food crop cassava with the aim of engineering resistance to African cassava mosaic virus, a member of a widespread and important family (Geminiviridae) of plant-pathogenic DNA viruses. Results Our results show that the CRISPR system fails to confer effective resistance to the virus during glasshouse inoculations. Further, we find that between 33 and 48% of edited virus genomes evolve a conserved single-nucleotide mutation that confers resistance to CRISPR-Cas9 cleavage. We also find that in the model plant Nicotiana benthamiana the replication of the novel, mutant virus is dependent on the presence of the wild-type virus. Conclusions Our study highlights the risks associated with CRISPR-Cas9 virus immunity in eukaryotes given that the mutagenic nature of the system generates viral escapes in a short time period. Our in-depth analysis of virus populations also represents a template for future studies analyzing virus escape from anti-viral CRISPR transgenics. This is especially important for informing regulation of such actively mutagenic applications of CRISPR-Cas9 technology in agriculture.

Improved crops can contribute to a world without hunger, if properly managed A world without hunger is possible but only if food production is sustainably increased and distributed and extreme poverty is eliminated. Globally, most of the poor and undernourished people live in rural areas of developing countries, where they depend on agriculture as a source of food, income, and employment. International data show a clear association between low agricultural productivity and high rates of undernourishment ( 1 ). Global studies have also shown that rapid reduction of extreme poverty is only possible when the incomes of smallholder farmers are increased ( 2 ). Therefore, sustained improvement in agricultural productivity is central to socioeconomic development. Here, we argue that with careful deployment and scientifically informed regulation, new plant breeding technologies (NPBTs) such as genome editing will be able to contribute substantially to global food security.

Recent advances in genome engineering (GE) has made it possible to precisely alter DNA sequences in plant cells, providing specifically engineered plants with traits of interest. Gene targeting efficiency depends on the delivery-method of both sequence-specific nucleases and repair templates, to plant cells. Typically, this is achieved using mediated transformation or particle bombardment, both of which transform only a subset of cells in treated tissues. The alternate approaches, stably integrating nuclease-encoding cassettes and repair templates into the plant genome, are time consuming, expensive and require extra regulations. More efficient GE reagents delivery methods are clearly needed if GE is to become routine, especially in economically important crops that are difficult to transform. Recently, autonomously replicating virus-based vectors have been demonstrated as efficient means of delivering GE reagents in plants. Both DNA viruses ( and ) and RNA virus ( ) have demonstrated efficient gene targeting frequencies in model plants ( ) and crops (potato, tomato, rice, and wheat). Here we discuss the recent advances using viral vectors for plant genome engineering, the current limitations and future directions.

Plant viruses infect many economically important crops, including wheat, cotton, maize, cassava, and other vegetables. These viruses pose a serious threat to agriculture worldwide, as decreases in cropland area may cause production to fall short of that required to feed the increasing world population. Under these circumstances, conventional strategies can fail to control rapidly evolving and emerging plant viruses. Genome-engineering strategies have recently emerged as promising tools to introduce desirable traits in many eukaryotic species, including plants. Among these genome engineering technologies, the CRISPR (clustered regularly interspaced palindromic repeats)/CRISPR-associated 9 (CRISPR/Cas9) system has received special interest because of its simplicity, efficiency, and reproducibility. Recent studies have used CRISPR/Cas9 to engineer virus resistance in plants, either by directly targeting and cleaving the viral genome, or by modifying the host plant genome to introduce viral immunity. Here, we briefly describe the biology of the CRISPR/Cas9 system and plant viruses, and how different genome engineering technologies have been used to target these viruses. We further describe the main findings from recent studies of CRISPR/Cas9-mediated viral interference and discuss how these findings can be applied to improve global agriculture. We conclude by pinpointing the gaps in our knowledge and the outstanding questions regarding CRISPR/Cas9-mediated viral immunity.

The first decade of genetic engineering primarily focused on quantitative crop improvement. With the advances in technology, the focus of agricultural biotechnology has shifted toward both quantitative and qualitative crop improvement, to deal with the challenges of food security and nutrition. Potato ( L.) is a solanaceous food crop having potential to feed the populating world. It can provide more carbohydrates, proteins, minerals, and vitamins per unit area of land as compared to other potential food crops, and is the major staple food in many developing countries. These aspects have driven the scientific attention to engineer potato for nutrition improvement, keeping the yield unaffected. Several studies have shown the improved nutritional value of potato tubers, for example by enhancing 1 seed protein content, vitamin C content, β-carotene level, triacylglycerol, tuber methionine content, and amylose content, etc. Removal of anti-nutritional compounds like steroidal glycoalkaloids, acrylamide and food toxins is another research priority for scientists and breeders to improve potato tuber quality. Trait improvement using genetic engineering mostly involved the generation of transgenic products. The commercialization of these engineered products has been a challenge due to consumer preference and regulatory/ethical restrictions. In this context, new breeding technolgies like TALEN (transcription activator-like effector nucleases) and CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated 9) have been employed to generate transgene-free products in a more precise, prompt and effective way. Moreover, the availability of potato genome sequence and efficient potato transformation systems have remarkably facilitated potato genetic engineering. Here we summarize the potato trait improvement and potential application of new breeding technologies (NBTs) to genetically improve the overall agronomic profile of potato.

Circular DNA is ubiquitous in nature in the form of plasmids, circular DNA viruses, and extrachromosomal circular DNA (eccDNA) in eukaryotes. Sequencing of such molecules is essential to profiling virus distributions, discovering new viruses and understanding the roles of eccDNAs in eukaryotic cells. Circular DNA enrichment sequencing (CIDER-Seq) is a technique to enrich and accurately sequence circular DNA without the need for polymerase chain reaction amplification, cloning, and computational sequence assembly. The approach is based on randomly primed circular DNA amplification, which is followed by several enzymatic DNA repair steps and then by long-read sequencing. CIDER-Seq includes a custom data analysis package (CIDER-Seq Data Analysis Software 2) that implements the DeConcat algorithm to deconcatenate the long sequencing products of random circular DNA amplification into the intact sequences of the input circular DNA. The CIDER-Seq data analysis package can generate full-length annotated virus genomes, as well as circular DNA sequences of novel viruses. Applications of CIDER-Seq also include profiling of eccDNA molecules such as transposable elements (TEs) from biological samples. The method takes ~2 weeks to complete, depending on the computational resources available. Owing to the present constraints of long-read single-molecule sequencing, the accuracy of circular virus and eccDNA sequences generated by the CIDER-Seq method scales with sequence length, and the greatest accuracy is obtained for molecules <10 kb long. Size-selected and amplified circular DNA molecules are sequenced on the PacBio platform and processed with a custom pipeline, resulting in full-length annotated genomes of circular DNA viruses and sequences of extrachromosomal circular DNA at single-molecule resolution.

Cotton leaf curl disease (CLCuD) is the major biotic constraint to cotton production on the Indian subcontinent, and is caused by monopartite begomoviruses accompanied by a specific DNA satellite, Cotton leaf curl Multan betasatellite (CLCuMB). Since the breakdown of resistance against CLCuD in 2001/2002, only one virus, the “Burewala” strain of Cotton leaf curl Kokhran virus (CLCuKoV-Bur), and a recombinant form of CLCuMB have consistently been identified in cotton across the major cotton growing areas of Pakistan. Unusually a bipartite isolate of the begomovirus Tomato leaf curl virus was identified in CLCuD-affected cotton recently. In the study described here we isolated the bipartite begomovirus Tomato leaf curl New Delhi virus (ToLCNDV) from CLCuD-affected cotton. To assess the frequency and geographic occurrence of ToLCNDV in cotton, CLCuD-symptomatic cotton plants were collected from across the Punjab and Sindh provinces between 2013 and 2015. Analysis of the plants by diagnostic PCR showed the presence of CLCuKoV-Bur in all 31 plants examined and ToLCNDV in 20 of the samples. Additionally, a quantitative real-time PCR analysis of the levels of the two viruses in co-infected plants suggests that coinfection of ToLCNDV with the CLCuKoV-Bur/CLCuMB complex leads to an increase in the levels of CLCuMB, which encodes the major pathogenicity (symptom) determinant of the complex. The significance of these results are discussed.

The first epidemic of cotton leaf curl disease (CLCuD) in early 1990’s in the Indian subcontinent was associated with several distinct begomoviruses along with a disease-specific betasatellite. Resistant cotton varieties were introduced in late 1990’s but soon resistance was broken and was associated with a single recombinant begomovirus named Burewala strain of Cotton leaf curl Kokhran virus that lacks a full complement of a gene encoding a transcription activator protein (TrAP). In order to understand the ongoing changes in CLCuD complex in Pakistan, CLCuD affected plants from cotton fields at Vehari were collected. Illumina sequencing was used to assess the diversity of CLCuD complex. At least three distinct begomoviruses characterized from the first epidemic; Cotton leaf curl Multan virus , Cotton leaf curl Kokhran virus and Cotton leaf curl Alabad virus , several distinct species of alphasatellites and cotton leaf curl Multan betasatellite were found associated with CLCuD. These viruses were also cloned and sequenced through Sanger sequencing to confirm the identity of the begomoviruses and that all clones possessed a full complement of the TrAP gene. A new strain of betasatellite was identified here and named CLCuMuB Veh . The implications of these findings in efforts to control CLCuD are discussed.