Combating HLB with gene editing

CRISPR/Cas9-mediated gene editing is a molecular technique that can be used to disrupt or eliminate the function of a specified gene (amongst other capabilities). In plant biology it has considerable advantages over traditional breeding approaches for crop improvement as it can be much faster in terms of generating new cultivars and, due to its pinpoint precision, reduces linkage drag that can inadvertently introduce unwanted traits into a particular cultivar.

The CRISPR/Cas9 system consists of a protein-RNA complex composed of two components; a single guide RNA (sgRNA) and the Cas9 endonuclease. The sgRNA provides specificity to the Cas9-sgRNA complex by targeting the endonuclease to a single DNA locus in the genome using RNA:DNA pairing. Therefore, by designing the appropriate site-specific sgRNAs, Cas9 can be targeted to any gene in the genome to induce a double-stranded DNA (dsDNA) break. These dsDNA breaks are most often repaired by an error-prone DNA repair pathway that can randomly create small insertions or deletions. This makes CRISPR/Cas9 useful for knocking out the function of a gene, as the targeted insertions and deletions that are created in the repair process usually change the reading frame of a gene, which in turn prevents the production of a functional protein.

Selection of potential HLB susceptibility genes

In response to the large detrimental impact HLB is having on the Citrus industry many groups within the Citrus research community have described how Citrus trees respond to HLB at the molecular level. This has generated a large number of global genomic datasets, an invaluable resource in future efforts to understand and combat HLB. However the use of differing technologies, reference genomes and reporting formats has previously prevented leveraging this wealth of information together as a whole.

Recently we have surveyed the literature and identified 28 publications documenting 71 such datasets. These include both transcriptomic and proteomic approaches and describe a diverse range of samples taken from different tissues, stages of HLB progression and Citrus species. Differentially abundant genes and proteins were manually extracted from the publications and collated. Then to make meaningful comparisons possible, phylogenetic orthology inference was used to identify orthologous genes between reference genomes. This allows the straight-forward translation of genes and/or proteins into one common genome annotation which facilitates meaningful comparison between genomic datasets. This analysis classified 27,855 C. sinensis v2.0 genes into 23,017 orthogroups and a total of 22,887 unique genes were identified as differentially abundant in at least one dataset (82% of the total genes).

We will utilise this analysis as a starting point to select potential HLB susceptibility genes by prioritising genes which are identified as differentially abundant in multiple studies. These genes are more likely to be biological responses of the Citrus tree to HLB. For instance, there are 1,029 differentially abundant genes in 10 or more datasets representing 4.65% of the total differentially abundant genes. The subsetted data includes many genes previously implicated in HLB susceptibility. Gene Ontology enrichment analysis identifies carbohydrate metabolism and defense response genes over-represented in the list, consistent with what is understood about the biology of HLB.

We will also consider biologically relevant subsets of the datasets, previously identified candidate genes, parologous genes and other candidates suggested by the community.

Generation of mutant population

To inactivate 1,200 potantial HLB-susceptibility genes, we will use a multiplex CRISPR/Cas9 approach. Our strategy will be to include four sgRNA genes targeting four different Citrus genes in each binary vector. This multiplex approach will therefore require a minimum of 300 constructs each containing a different set of four sgRNAs, for a total of 1,200 gene knockouts in 300 Citrus plants. We intend to create and maintain four independent Citrus transgenic plants for each set of four sgRNAs, so the final plant collection created will consist of 1,200 plants housed at the Yale Plant Facility.

The combination of each set of sgRNAs will be determined by their phylogenetic relationship to one another and location in the genome. Their patterns of co-expression and predicted function will also be considered. The ortholog analysis we have done (see above) will also be used such that sgRNA targeting genes within the same orthogroup will be included on the same binary vector to overcome functional redundancy between paralog genes. This approach will ensure that genetic redundancy does not mask a potential phenotype of interest (i.e. HLB resistance) when this transgenic Citrus collection is used for screening.

Agrobacterium-mediated transformation of Citrus sinensis cv Valencia will be carried out based on our optimized protocols. Selection of transgenic plants expressing high levels of Cas9 and sgRNAs is facilitated by the use of the GFP-NPTII bifunctional selection marker. We will maximize the mutation rates in the selected Citrus plants by exposing edited plants to repeated heat stress treatments, which we have shown to drastically increase the editing rate in plants.

We will rapidly screen at least 12 transformants for each construct and select two robust lines, in which editing has been demonstrated to be at or close to 100% in the four targeted genes, to be maintained. To precisely and rapidly measure gene editing rates in the selected plants we will employ targeted amplicon sequencing. Information regarding the mutation level at each locus in each transgenic plant will be provided on the project website.

Survey of consumer acceptance and economic viability of edited Citrus

The potential application of using CRISPR/Cas9-mediated editing to develop HLB resistance is constrained by whether consumers will accept CRISPR/Cas9-edited citrus. If consumers will not accept this type of citrus then growers will not grow it. Much research has been conducted on consumer acceptance of genetically modified foods, and typically consumers are willing to pay more to avoid this option. However, a number of different factors are found to influence acceptance including the type of product, the location of the consumer, the benefit conferred by the modification and the technology itself. It is important that we learn about consumer reactions so that when such products become available to consumers we can assess what are the critical factors that determine acceptance. This is both necessary to determine demand (and profitability if such products are introduced) and to develop an understanding of how to speak to consumers about complex technologies.

To aid in this process we will convene consumer focus groups to discuss the use of CRISPR/Cas9 (and other forms of technology) in food production. The focus of the discussions will be on consumer reaction to information about technology, including why it is used, how it is used, and how it may be regulated or labeled. This will provide a deeper understanding about the key concepts that concern or relieve concern about the use of complex technology in food consumption. Infomation from these discussions will also help shape a larger, representative consumer survey to test messages that successfully alleviate concerns about technology on a larger scale. In addition, the survey can incorporate methods related to valuing food products to determine the impact of concern on willingness to pay for food products (e.g. orange juice) produced with different technologies.

From this work a white paper will be developed and made available that provides input to researchers on how to communicate effectively about CRISPR with consumers. It will also shed light on the economic implications of this project by identifying barriers to adoption of CRISPR-edited citrus by growers and estimating the economic importance of these barriers.

In recent years the Irish and Jacob labs have developed approaches that have significantly improved CRISPR/Cas9 efficiency in citrus. These include the use of codon-optimized versions of Cas9, along with specific promoters that augment Cas9 activity in dividing cells. The groups have also developed vectors to facilitate identification of transgenic events by using a combination of selectable markers (GFP fluorescence and resistance to kanamycin) and optimized citrus transformation protocols to generate more citrus transgenic plants per transformation. Research from these collaborating labs has also led to advances which increase the rate of CRISPR/Cas9-mediated mutagenesis by extending the time of the citrus regeneration process and the exposure to heat stress which increases the frequency of CRISPR/Cas9-induced mutations many-fold relative to transformed plants not exposed to heat stress.

The labs have demonstrated that multiplex gene editing is feasible in Citrus. Multiplex gene editing refers to the targeting of multiple genes in a single event, so as to abrogate the function of several genes at once. This was utilized to successfully target three thorn development genes simultaneously using multiplex CRISPR/Cas9. This multiplex approach was extremely valuable in addressing genetic redundancy between the thorn developmental genes of Citrus as only by mutating several genes simultaneously was it possible to uncover overlapping genetic functions that were not apparent in single knockout lines. As genetic redundancy could also play a role in HLB susceptibility, the ability to inactivate multiple genes at once will be essential in testing the roles of multiple genes in disease response. Thus the labs involved in this project have a robust protocol to rapidly, effectively, and efficiently knock out multiple genes simultaneously in Citrus.

The CRISPR citrus project to identify HLB susceptibility genes in a Citrus population generated using multiplexed CRISPR/Cas9 gene editing is funded as part of the USDA-NIFA’s Emergency Citrus Disease Research & Extension (ECDRE) program.

This program promotes collaboration, open communication and the exchange of information to accelerate the application of scientific discovery and technology to farm-level solutions for HLB.