Progress 06/15/20 to 06/14/24
Outputs
Target Audience:The target audiences for the duration of this project include graduate students and postdocs as well as plant pathologists, mycologists, molecular biologists and genome engineers in academia, industry and government agencies. Changes/Problems:We encountered technical difficulties using cytosine base editor for targeted mutagenesis and the bacterial retron for on-site delivery of DNA repair templateas initially proposed for Objectives 3 and 4. As a result, the advancedprime editing technology has been used as an alternative approach to complete Objectives 3 and 4. Using prime editing, we successfully achieved base substitution, deletion and insertion (for targeted mutagenesis, allele conversion and epitope tagging) as well as on-site delivery of DNA repair templateinthe rice blast fungus. What opportunities for training and professional development has the project provided?The project provided training and professional development opportunities to one postdoc (Matthew Wheatley), two graduate students (Justin Shih and Yang Hsu) and a research technologist for performing and studying gRNA spacer prediction, fungal transformation and CRISPR/Cas genome editing technologies. This project also offered learning and educational opportunities to graduate students via the PPATH 597 Genome Editing course. How have the results been disseminated to communities of interest?The results and findings from this project were disseminated to communities of interest through research seminars as well as oral and poster presentations at national and international conferences. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Genome-wide prediction of Cas9 gRNA spacers has been performed for genomes of plant pathogenic fungi. Strikingly, over 90% of the gRNA spacers predicted across these fungal genomes belong to the highly specific classes of gRNA spacers that would enable genome-wide targetability. However, fungal genome data in Ensembl and other databases have variable annotations and sequence qualities and are often presented with different formats. These issues present challenges for us to generate consistent databases for genome-wide gRNA spacer prediction. Multiplex genome editing has been successfully achieved in Magnaporthe oryzae using the polycistronic tRNA-gRNA approach, leading to the efficient generation of single, double and triple gene knockout mutants. In absence of donor templates, Cas9-induced double strand breaks trigger fungal cell death or genomic rearrangement. By promptly providing donor DNA templates, however, precise genome editing based on homology-dependent repair was achieved at high efficiencies (up to over 90%). It was noticed that the CRISPR/Cas-mediated editing frequencies in M. oryzae also depended on genomic location of target sites and was likely influenced by flanking repetitive sequences and transposable elements. A manuscript titled "CRISPR/Cas9-enabled multiplex genome editing and locus-dependent DNA repair in Magnaporthe oryzae" has been prepared for publication. At least three attempts have been made to create targeted gene mutation without double strand breaks using a cytosine base editor. Although some fungal mutants were obtained, DNA sequence analysis has shown that none of these mutants were resulting from precise base editing. In addition, we failed to achieve genome editing in M. oryzae with the bacterial retron method after repeated attempts. Instead of using cytosine base editor for CRISPR Stop or using the bacterial retron for precise editing, we decided using the advanced prime editing technology for targeted mutagenesis and precise editing (base substitution, deletion and insertion) in M. oryzae without DNA double strand breaks. Because prime editing utilizes the reversely transcribed cDNA from prime editing gRNA (pegRNA) as the repair template, the approach allows onsite delivery of donor DNA template for efficient genome editing. As a result, exogenous DNA repair templates do not need to be co-delivered into M. oryzae. Our study has shown 67-100% editing efficiencies for 2 bp base substitutions in two effector genes Nep1-like 1 and Nep1-like 3 (NLP 1 and NLP3) in M. oryzae using the dual-pegRNA prime editing strategy. Furthermore, 3 bp insertions and deletions in the NLP1 gene were achieved at 55% efficiency, whereas 24 bp insertion and deletions were obtained at 9.1% efficiency. With its versatility and editing efficiency, prime editing technology should significantly facilitate the precise and multiplex genome editing in rice blast and other fungal pathogens. A manuscript titled "Precise genome manipulation in the rice blast fungus with prime editing" has been prepared for publication.
Publications
|
Progress 06/15/22 to 06/14/23
Outputs
Target Audience:The target audiences during this reporting peroid include graduate students and postdocs as well as plant pathologists, molecular biologists and breeders in academia, industry and government agencies. Changes/Problems:We previously encountered technical difficultiesusingcytosine base editing and bacterial retron editing approaches as initially proposed forGoals 3 and 4. By using the advanced and versatile prime editing technology, we successfully achieved base substitution, deletion and insertion as well as on-site delivery of DNA repair templates in the rice blast fungus. What opportunities for training and professional development has the project provided?The project provided training and professional development opportunities to two graduate students (Justin Shih and Yang Hsu) and a research technologistfor them to learn the fungal transformation and genome editing technologies. This project also offered learning and educational opportunities to graduate students via the PPATH 597 Genome Editing course. How have the results been disseminated to communities of interest?The results and findings from this project were disseminated to communities of interest through research seminars as well as oral and poster presentations at national and international conferences. What do you plan to do during the next reporting period to accomplish the goals?We plan to further optimize prime editing and multiplex genome editing techniques for the rice blast fungus in the next reporting period. I will continue to teach PPATH 597 genome editing class (3 credits) andplan to submit two manuscripts related to multiplex genome editing and prime editing of M. oryzae.
Impacts
What was accomplished under these goals?
During this reporting period, we have focused on achieving Goals 3 and 4. Instead of using cytosine base editor and CRISPR-Stop strategy, we used the advance prime editing technology to successfully performtargeted mutagenesis (base substitution, deletion and insertion) without DNA double strand breaks (Goal 3). The prime editing approach also allows on-site delivery of donor DNA template for efficient and precise genome editing (Goal 4). Because prime editing utilizes the reversely transcribed cDNA from prime editing gRNA (pegRNA) as the repair template, exogenous DNArepair templates do not need to be produced and codelivered into the rice blast fungus (Magnaporthe oryzae). Our study has shown 67-100% editing efficiencies for 2 bp base substitutionsin two effector genes Nep1-like 1 and Nep1-like 3 (NLP 1 and NLP3) in M. oryzaeusing the dual-pegRNA prime editing strategy. Furthermore, 3 bp insertions and deletions in the NLP1 genewere achieved at 55% efficiency, whereas24 bp insertion and deletions were obtained at 9.1% efficiency. With suchversatile and efficient editing inM. oryzae, thisprime editing technology will faciltate the studies of virulence mechanisms and functional genomics of rice blast and other fungal pathogens.
Publications
|
Progress 06/15/21 to 06/14/22
Outputs
Target Audience:The target audiencesinclude graduate students and postdocs as well as plant pathologists, molecular biologists and breeders in academia, industry and government agencies. Changes/Problems:We encountered problems with cytosine base editing and bacterial retron editing approaches outlined in Goals 3 and 4. We plan to use a new base editor system and replace the bacterial retron approach with the prime editing method in our future experiments. Thisproject has beensignificantly deIayed due to thedifficulties to recruit qualified personnelto carry out the proposed research activities. As a result, I requested an extension of this project, which has been approved by USDA/NIFA. What opportunities for training and professional development has the project provided?This project provided training and professional development opportunities to a postdoctoral research associate (Matthew Wheatley) for studying CRISPR/Cas-mediated genome editing technologies. This project also offered learning and educational opportunities to graduate students via the PPATH 597 Genome Editing course. How have the results been disseminated to communities of interest?The results and findings from this project were disseminated to communities of interest through the journal article as well as meeting and seminar presentations. What do you plan to do during the next reporting period to accomplish the goals?We have identified problems associated with cytosine base editing and bacterial retron editing. We will modify andoptimizecytosine base editors for efficientCRISPR-Stop mutagenesis. We also plan toreplace the bacterial retron approach with the prime editing methodfor precise genome editing in M. oryzae.I will continue toteach PPATH 597 genome editing class (3 credits) in Fall 2022 andplan tosubmit one or two manuscripts in the next reporting period.
Impacts
What was accomplished under these goals?
Goal 1. Genome-wide prediction of Cas9 gRNA spacers has been performed for 28 genomes of plant pathogenic fungi. However, fungal genome data in Ensembl and other databases have variable annotations and sequence qualities and are often presented with different formats. These issues present challenges for us to generate consistent databases for genome-wide gRNA space prediction. Goal 2. Multiplex genome editing has been successfully achieved in Magnaporthe oryzae using the polycistronic tRNA-gRNA approach, leading to the efficient generation of single, double and triple gene mutants. In absence of donor templates, Cas9-induced double strand breaks trigger fungal cell death or genomic rearrangement. By promptly providing donor templates, however, precise genome editing based on homology-dependent repair wasachieved athigh efficiencies.We noticed that the CRISPR/Cas-mediated editing frequencies in M. oryzae also dependedon genomic location of target sites and was likelyinfluenced by flanking repetitive sequences and transposable elements. A manuscript has been completed and will be submit to a refereed journal soon. Goal 3. At least three attempts have been made to createtargeted gene mutation without double strand breaks using a cytosine base editor. Although some fungal mutants were obtained, DNA sequence analysis has shown that none of these mutants were resulting from precise base editing. We have identified the problems and will modify the cytosine base editorfor CRISPR-Stop mutation in M. oryzae. Goal 4. Two genome editing experiments were performed in M. oryzae using a bacterial retron. However, we were unable to achieve precise editing using this method. Recently, we have suceeded in using prime editors for precise editing of rice genes. We decided to test theprime editing methodfor precise editing of M. oryzae genes in our future experiments.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Wheatley, M.S. and Yang, Y. 2021. Versatile applications of the CRISPR/Cas toolkit in plant pathology and disease management. Phytopathology 111:10801090.
|
Progress 06/15/20 to 06/14/21
Outputs
Target Audience:The target audiences reached during the reporting period include students and postdocs as well as plant pathologists, molecular biologists and breeders in academia, industry and government agencies. Changes/Problems:Due to the variable sequence qualitiesand different annotation formats of fungal genomes, we encountered difficulties to generate consistent database for genome-wide prediction of gRNA spacers. As a result, we may have to reduce the number of fungal genomes and focus on high-quality genome sequences that have consistent annotation formats for gRNA spacer prediction. Due to the pandemic impact, I encountered difficulties to recruit and retain qualified personnel to carry out the project. It is likely that I will request an extension of this project due to the delay of completing the proposed goals. What opportunities for training and professional development has the project provided?This project provided training and professional development opportunities to a postdoctoral research associate(Matthew Wheatley) for studying CRISPR/Cas-mediatedgenome editing technologies. This project also offered learning andeducational opportunities to graduate students via the PPATH 597 Genome Editing course. How have the results been disseminated to communities of interest?The results and findings from this project have been disseminated to communities of interest through journal article, conference presentation andresearch seminar. What do you plan to do during the next reporting period to accomplish the goals?We will improve bioinformatics tools to overcome the challenges associated with genome-wide prediction of specific gRNA spacers in plant pathogenic fungi. We will also optimize CRISPR/Cas editing constructs to resolve technical issues associated with using cytosine base editor and bacterial retron for CRISPR-Stop mutation and efficient HDR editing, respectively. In addition, Iplan to teach PPATH 597 genome editing again andsubmit one or two manuscripts for publication in refereed journals during the next reporting period.
Impacts
What was accomplished under these goals?
Goal 1. Genome-wide prediction of Cas9gRNA spacers has been performed for 28genomes of plant pathogenic fungi. However, fungal genome datain Ensembl and other databases have variable annotations and sequence qualities and are often presented with differentformats. These issuescreate challenges for us to generate consistent databases for genome-wide gRNA space prediction. Goal 2. Multiplex genome editing has been successfully achieved in Magnaporthe oryzaeusing the polycistronic tRNA-gRNA approach, leading to the efficient generation of single, double and triple gene mutants. We noticed that the CRISPR/Cas-mediated editingfrequencies inM. oryzaeappear to be dependent on genomic location of target sites and are likely influenced by flanking repetitive sequences and transposable elements. Based on the experimental results, amanuscript has been prepared for submission to a refereed journal. Goal 3. At least three attempts have been made to achieve targeted gene mutation without double strand breaks using a cytosine base editor. Although some fungal mutants were obtained, DNA sequence analysis has shown thatnone of these mutants were resulting from precise base editing. As a result, further optimization of cytosine base editors is required for successful CRISPR-Stop mutation in M. oryzae. Goal 4. Two editing experiments were performed using a bacterial retron. However, we were unable to achieveefficient HDR editing using this method. We need to optimize the gene constructs based on the bacterial retron and improve the retron-based HDR editing in M. oryzae during the second year of this project.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Wheatley, M.S. and Yang, Y. 2020. Versatile applications of the CRISPR/Cas toolkit in plant pathology and disease management. Phytopathology (doi.org/10.1094/PHYTO-08-20-0322-IA, published as First Look in Dec. 2020)
|
|