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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.

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.

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.

Lack of critical assessment and responsible reporting of proof-of-concept agricultural biotechnologies such as CRISPR–Cas can delay innovation, jeopardize public trust and waste resources, especially in the Global South. In this commentary, we propose solutions to facilitate a more responsible innovation pipeline and to realize the potential of biotechnology in agriculture.Mehta and Vanderschuren advocate for more stringent standards in reporting agricultural biotechnology research.

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.

Ending all forms of hunger by 2030, as set forward in the UN-Sustainable Development Goal 2 (UN-SDG2), is a daunting but essential task, given the limited timeline ahead and the negative global health and socio-economic impact of hunger. Malnutrition or hidden hunger due to micronutrient deficiencies affects about one third of the world population and severely jeopardizes economic development. Staple crop biofortification through gene stacking, using a rational combination of conventional breeding and metabolic engineering strategies, should enable a leap forward within the coming decade. A number of specific actions and policy interventions are proposed to reach this goal. Biofortification is an effective means to reduce micronutrient malnutrition. Here, the authors review recent advances in biofortification and propose stacking multiple micronutrient traits into high-yielding varieties through the combination of conventional breeding and genetic engineering approaches.

Cassava is an important staple crop that provides food and income for about 700 million Africans. Cassava productivity in Africa is limited by viral diseases, mainly cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Genetic barriers such as high heterozygosity, allopolyploidy, poor seed set, and irregular flowering constrain the development of virus-resistant cassava varieties via conventional breeding. Genetic transformation represents a valuable tool to circumvent several challenges associated with the development of virus resistance and other valuable agronomic traits in cassava. The implementation of genetic transformation in many local African cultivars is limited either by the difficulty to produce friable embryogenic callus (FEC), low transformation, and/or regeneration efficiencies. Here, we report the successful induction of organized embryogenic structures (OES) in 11 farmer-preferred cultivars locally grown in Ghana. The production of high quality FEC from one local cultivar, ADI 001, facilitated its genetic transformation with high shoot regeneration and selection efficiency, comparable to the model cassava cultivar 60444. We show that using flow cytometry for analysis of nuclear ploidy in FEC tissues prior to genetic transformation ensures the selection of genetically uniform FEC tissue for transformation. The high percentage of single insertion events in transgenic lines indicates the suitability of the ADI 001 cultivar for the introduction of virus resistance and other useful agronomic traits into the farmer-preferred cassava germplasm in Ghana and Africa.

Cassava (Manihot esculenta) is a staple crop in sub-Saharan Africa threatened by several viral diseases. Here, we describe the genome sequence of a novel bipartite cheravirus (family Secoviridae) infecting cassava in the Democratic Republic of Congo and Tanzania. We designate the new virus “cassava Congo cheravirus”. Each RNA segment encodes a single polyprotein (P1 and P2 for RNA1 and RNA2, respectively), embedded with various putative cleavage sites (six and three in P1 and P2, respectively), consistent with members of the genus Cheravirus. We note two new features in the P1: (i) the presence of two domains, X1 and X2, upstream of the putative helicase region, which we also predict in other cheraviruses and (ii) the presence of a Maf/HAM1-like inosine triphosphatase (ITPase) domain, a rare motif among viruses only previously detected in three potyviruses and a torradovirus, all of which infect plants from the Euphorbia family. Phylogenetic analyses placed the virus firmly within the genus Cheravirus, with amino acid identities in the Pro-Pol and coat protein regions well below existing ICTV species thresholds, supporting its classification as a virus belonging to a new species in the Cheravirus genus. Spatially distinct isolates from Bas-Congo, South-Kivu, and Tanzania form three genetic clusters, with evidence of recombination in both RNA segments. These results expand the known diversity of cassava viruses and suggest possible adaptation to the cassava host via ITPase acquisition.

Extrachromosomal circular DNA (eccDNA) has been described in several eukaryotic species and has been shown to impact phenomena as diverse as cancer and herbicide tolerance. EccDNA is thought to arise mainly through transposable element (TE) mobilization. Because studies based on short-read sequencing cannot efficiently identify full-length eccDNA forms generated from TEs, we employed the CIDER-Seq pipeline based on long-read sequencing, to obtain full-length eccDNAs from Arabidopsis . The generated eccDNA datasets identified centromeric/pericentromeric regions as hotspots of eccDNAs with several eccDNA molecules originating from Helitron and LTR TEs. To investigate the role of epigenetic marks on TE-derived eccDNA biogenesis, we studied Arabidopsis methylation mutants dcl3 , rdr6 , ros1 , and ddm1 . Contrasting the TE-suppression previously reported in the hypermethylated ros1 mutants, we identified activation of TEs in ros1 , specifically of LTR/Gypsy TEs. An enrichment of LTR/Copia elements was identified in actively dividing calli and the shoot apical meristem (SAM). Uncharacterized “variable TEs” with high eccDNA and expression were identified in the SAM, including ATCOPIA58 . Together, our study reveals the genomic origins of eccDNAs and delineates the link between epigenetic regulation, transposon mobilization, and eccDNA biogenesis.