09/12/2022
FRA Developing Genome Editing to Confer HWA Resistance
FRA continues to address the loss of a $10-$15 million annual farm income to the NC nursery and landscape industry, and the loss of native hemlock stands because of an invasive pest, the hemlock woolly adelgid (HWA), into western North Carolina. FRA is exploiting resistance in native eastern and Carolina hemlocks to help re-establish hemlock as a valuable nursery and landscape crop, and to restore natural hemlock stands impacted by HWA-induced decline and mortality. Recently developed genome editing technologies offer enormous potential for advancing the genetic improvement of hemlocks for HWA resistance/tolerance. Editing genomic DNA to induce loss- or gain-of-function mutation enables precise heritable improvement of phenotypic traits, thereby providing a powerful tool for hemlock restoration. Genome editing can induce novel traits such as pest resistance/tolerance that is not available in natural populations, making it a particularly attractive approach for targeting invasive pests and pathogens. The lack of sufficient genomic and biotechnological tools is a serious limitation for genetic and functional genomic studies to improve the health of hemlock trees. Biotechnology-enabled genetic/genomic research and development of robust genome editing systems would accelerate the production of robust hemlock variants. The Forest Biotech Group at NC State University is experienced in developing CRISPR technologies in forest trees. We have recently developed a robust method for the expression, purification, and assembly of functional CRISPR machineries in vitro and have demonstrated their genome editing activity. We will use our established genome editing platforms to determine the most effective approach for delivery of CRISPR machineries into hemlock trees for in vivo editing. Our genome editing approach should produce highly stable, predictable, non-GMO, and heritable forms of genetic improvements in hemlocks, which translates to reliable improvements in phenotypic traits.
To generate the CRISPR-Cas machineries necessary for transgene-free editing of hemlock somatic embryogenic (SE) lines, we expressed and purified functional CRISPR- Cas proteins for SpCas9, LbCas12a, and AsCas12a and demonstrated the efficiency and specificity of their in vitro cleavage of target DNA sequences. These recombinant proteins were used for assembly of functional Cas-gRNA ribonucleoprotein (RNP) complexes targeting the genome editing of hemlock SE lines. We have produced enough Cas proteins (>5 mg) for high throughput editing of hemlock SE cultures. To test the CRISPR system in hemlock SE lines, we designed an in vitro validation of gRNAs to target hemlock phytoene desaturase (PDS) gene. To deliver the CRISPR-RNP complexes into hemlock SE, we have developed a novel protocol for isolation and transfection of hemlock SE-originated protoplasts. This result confirmed that we have successfully developed a platform for the delivery of CRISPR-RNP complexes into hemlock protoplasts. These RNP complexes were then delivered into hemlock protoplast cells for evaluating CRISPR-related mutational events through Next Generation Sequencing (NGS). CRISPR-driven mutation of hemlock is continuing to be evaluated and modified.
FRA has been collecting potentially resistant hemlock cuttings throughout the range of hemlocks for use in developing genome editing technology. The cuttings collected in 2019 and 2020 from eastern hemlocks that survived the rooting process have grown well since coming out of the rooting beds. The best plants from these cohorts play a key role in the current phase of the process to apply CRISPR in HWA resistance development – identifying targets for genome editing. HWA-infested and non-infested copies of various clones, at several points in the development of HWA, are phenotypically assessed and tissue sampled. RNA sequencing of the tissue samples will help us understand how genes are being expressed in the presence and absence of HWA, guiding the genome editing phase. In addition to the CRISPR project, the HWA screening study initiated in 2017 is ongoing. It has generated phenotypic data, but generating significant effects from adelgid infestation has remained elusive, at least in part due to lower than desired levels of adelgid settling and survival to date. We are also growing putative hybrids produced by 2019 breeding efforts, some of which should be three-way hybrids and hybrid back-crosses (pending molecular verification), as well as two half-sib families from several of the same Carolina hemlock maternal parents as the hybrids. The half-sib families will be utilized as check lots in screening trials as we should have stable, long-term access to the parents for seed collection indefinitely, giving us a consistent genetic comparison among trials over time. We will add additional three-way and back-cross hybrids produced by 2021 breeding efforts. These hybrids have good potential for resistance and could be released as horticultural cultivars in their current form if they have suitable growth characteristics, but they would lack the diversity and similarity to native hemlocks needed for restoration of forest stands. Breeding in 2021 also included potential hybrid crosses between eastern hemlock and resistant exotic species, but we anticipate these hybridization attempts with an eastern hemlock parent will be our last effort of this type for at least several years, as all previous attempts of this type have been unsuccessful. Future breeding efforts in eastern hemlock will prioritize crosses within our population of resistance candidate genotypes.
