Improving Fungal Genome Editing And Plant Disease Diagnostics With Crispr Cas Technologies PDF Download

As a plant pathologist, I am interested in exploring new technologies to study plant-microbe interactions as well as improving crop disease management. Recently, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) system has emerged as a powerful technology with versatile applications in basic and applied research in medicine and agriculture. The goal of my dissertation project has been focused on improving and applying the CRISPR/Cas toolkit to facilitate efficient genome editing of fungal plant pathogens as well as sensitive and reliable diagnosis of plant diseases. The success of CRISPR/Cas9-based multiplex genome editing in the rice blast fungus and development of Cas12a-based diagnostics for citrus greening pathogen and phytoplasmas highlighted in my study demonstrate the utility and broad application of CRISPR/Cas technologies in plant pathology and disease management. The first objective of my research was to generate an online tool to aid in the design and selection of specific guide RNA (gRNA) spacers for genome editing of plant pathogens. The use of highly specific gRNAs is required to prevent unintended off-targeting effects. The CRISPR-PLANT v2 gRNA prediction pipeline was used for the genome-wide prediction of highly specific gRNA spacers for fifteen genomes of bacterial, oomycete, and fungal plant pathogens. Of the gRNAs predicted across these genomes, over 90% of the gRNA spacers belong to the highly specific classes of gRNA spacers and exhibit genome-wide targetability. The resulting gRNA spacer database and CRISPR-Pathogen webtool will be available to the plant pathology and microbiology research communities to facilitate the design of specific gRNAs and application of CRISPR/Cas9 genome editing in microbial plant pathogens. My second objective was to utilize the CRISPR/Cas9 system to improve the efficiency of single and multiplex genome editing in fungal plant pathogens. Magnaporthe oryzae is the causal agent of rice and wheat blast diseases and poses a major threat to rice and wheat production worldwide. As described in Chapter 3, CRISPR/Cas9-mediated multiplex genome editing in M. oryzae was successfully achieved using the polycistronic tRNA-gRNA (PTG) strategy. Upon creation of double stranded breaks (DSBs) by Cas9, targeted gene mutation in M. oryzae was created via either non-homologous end joining (NHEJ) or homology-directed repair (HDR) depending on the targeted loci. In absence of donor templates, Cas9-induced DSBs frequently triggered genomic rearrangement, leading to the loss of PCR-based amplification of target sites. By providing donor templates, however, HDR-mediated precise genome editing was achieved at the efficiency up to 100% when targeting a single locus. The PTG-based multiplex genome editing via HDR also successfully generated double and triple gene mutants. Interestingly, the HDR and NHEJ editing frequencies in M. oryzae appear to be dependent on genomic location of target sites and are likely influenced by flanking repetitive sequences and transposable elements. The resulting CRISPR/Cas9 tools and strategies from this study are expected to aid in the efficient genome editing and functional genomics analysis for M. oryzae and other fungal species. My third objective was to adapt the Cas12a-based method to enable highly sensitive and specific detection of pathogen nucleic acids for rapid and accurate diagnosis. As illustrated in Chapters 4 and 5, the citrus greening pathogen (Candidatus Liberibacter asiaticus) and purple potato top (PPT) phytoplasma were selected as target pathogens. The Cas12a-based DETECTR (DNA endonuclease-targeted CRISPR trans reporter) assay enabled highly specific and sensitive detection of CLas and Group VI phytoplasmas known to cause PPT from the infected samples. The DETECTR assay couples isothermal amplification and Cas12a trans-cleavage of fluorescent reporter oligos and enables detection of pathogen nucleic acids at the attomolar level. The DETECTR assay was able to accurately detect the presence of pathogen nucleic acids across all infected samples and was shown to be highly specific across closely related species. The improvement in detection sensitivity and flexibility of the DETECTR technology, positions the DETECTR assay as a suitable tool for early detection of pathogen nucleic acids. Furthermore, the DETECTR strategy allows flexibility to capture assay outputs with fluorescent microplate reader or lateral flow assay for potentially high-throughput and/or field-deployable disease diagnostics.