Source: IOWA STATE UNIVERSITY submitted to
A DATA-DRIVEN APPROACH TO CRISPR DESIGN FOR REDUCED OFF-TARGET ACTIVITY IN PLANT GENOME EDITING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1010354
Grant No.
2016-33120-25628
Project No.
IOW05480
Proposal No.
2016-06247
Multistate No.
(N/A)
Program Code
HX
Project Start Date
Sep 1, 2016
Project End Date
Aug 31, 2019
Grant Year
2016
Project Director
Wang, K.
Recipient Organization
IOWA STATE UNIVERSITY
2229 Lincoln Way
AMES,IA 50011
Performing Department
Agronomy
Non Technical Summary
Research described in this proposal addresses BRAG standard program area 5(d) "Research addressing off-target genotypic and/or phenotypic effects in GE organisms developed using genome editing technology."The process of genome editing seeks to make specific changes to DNA that improve crops through the addition or enhancement of agronomically important traits. The CRISPR-Cas9 (clustered regularly-interspaced short palindromic repeats with associated Cas9 protein) system accomplishes this well, but has been observed to make changes in non-target areas of genomes (off-target effects) as well. This can complicate research and interfere with trait development. The goal of this work is to improve CRISPR design to reduce off-target effects, thus enabling scientists to make changes only to specific genes and traits of interest. This will be accomplished as follows. In the design of CRISPRs, sequence-based bioinformatics tools are used to identify both target and off-target sites. Currently, bioinformatics predictions of off-target activities, especially for crop species, lack biological validation. This research seeks to generate experimental data that can be used to improve the predictive reliability of plant CRISPR specificity through the improvement of an open-source CRISPR design tool developed by Iowa State University. Improving predictive accuracy and reducing off-target mutation requires iterative, data-driven modifications to the algorithms underlying the functionality of existing CRISPR analysis tools. This will be accomplished by (1) evaluating high-fidelity enzymes for off-target cleavage reduction and (2) validating off-target activity in transgenic events. In addition, information about CRISPR technology and its robustness for targeted editing will be communicated to both research and regulatory communities. This research brings together extensive expertise in bioinformatics, biochemistry, plant genetic transformation, and risk assessment. This project's interdisciplinary team intends not only to improve the existing CRISPR design tool and system for plant genome editing, but also to generate important data addressing off-target genotypic effects in crop species such as maize and rice.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011530104050%
2011510104050%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1510 - Corn; 1530 - Rice;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
The goal of this proposal is to develop and improve a CRISPR design tool that can reduce mutation frequencies on unintended DNA sequences (off-target effects) in plant genome editing. Our specific objectives are: (1) to analyze and improve predictions made by a CRISPR design tool developed by Iowa State University with the goal to reduce off-target mutations; (2) to validate off-target cleavage and reduction by high-fidelity nucleases using an in vitro approach; (3) to validate off-target activity on transgenic events in planta; (4) to develop and carry out outreach and communication activities describing CRISPR technology and its robustness for targeted editing to both the research and regulatory communities.
Project Methods
Computational, biochemical, and molecular biological approaches will be used in objectives 1-3.Objective 1. Analyze and improve predictions made by a CRISPR design tool developed by Iowa State University with the goal to reduce off-target mutations (Year 1-3). For specific maize and rice genes, we will use the available software such as CGAT (designed by Iowa State University, a co-PD of this project), CHOP-CHOP, and CRISPR-P to identify putative target sites of interest as well as to predict potential off-target sites based on maize and rice reference genome analysis. We will review differences in sites identified among multiple tools, document accuracy of predictions based on experimental results, develop the improved algorithms for gRNAs design in plants, and deploy it via the CGAT tool site (http://cropbioengineering.iastate.edu/cgat/). We will also document differences between target and off-target prediction based on reference genome analysis and alternate germplasm experimentation, and report initial estimates of off-target changes that are not predicted based on reference genome analysis.Objective 2. Validate off-target cleavage and reduction by high-fidelity nucleases using an in vitro approach (Year 1-3). We will perform biochemical experiments to validate off-target sites and to evaluate the on-target activity and specificity of high-fidelity enzymes. We will test cleavage rates for targets and potential off-target sites to determine the expected relative rate of off-target editing. Wild-type (WT) Cas9 protein and high-fidelity variants will be expressed in E. coli and purified. For each target identified in Objective 1, we will construct a guide RNA (gRNA) template and both on-target and off-target DNA using oligonucleotide ligation into a vector. To form the Cas9-gRNA complex, we will incubate equimolar amounts of protein and RNA prior to initiation of cleavage reactions. Cleavage rates are determined using time-course assays. In addition, we will test cleavage for high-fidelity enzymes to determine whether on-target cleavage efficiency is maintained and off-target cleavage efficiency is decreased. This approach will enable rapid testing of potential off-target sites predicted by the CGAT design tool described in Objective 1. Optimal target sites and high-fidelity enzymes can be further tested in in vitro editing trials outlined in Objective 3.Objective 3. Validate off-target activity on transgenic events in planta (Year 1-3). For in planta work, we will choose target sequences and high-fidelity enzymes that identified and tested in Objective 1 and 2. These molecules will be introduced into corn and rice using genetic transformation methods such as Agrobacterium-mediated or biolistic-mediated method. We will first screen for herbicide resistant callus events for deletion mutations (edits) at the target gene site. Once the edited events are identified, we will then determine the Cas9 transgene copy numbers in these events using quantitative Polymerase Chain Reaction (qPCR). We will aim at producing 100 to 150 independent lines for each construct. Transformed corn and rice plants will be analyzed for both target and off-target mutational frequencies using targeted deep sequencing. Observed off-target changes will be analyzed to determine how best to adapt CRISPR design parameters used by each tool, with algorithm changes coded into the CGAT tool (based on accessibility of the code-base). These experiments will be repeated in both maize and rice for additional targets. Objective 4. Develop and carry out outreach and communication activities describing CRISPR technology and its robustness for targeted editing to both the research and regulatory communities (Year 1-3). We will organize a summer training workshop for K-14 educators and reach out to regulators and the general public through lectures and symposia. In a 4-day summer training workshop, learners will experience integrated learning streams with a critical core of immersive laboratory and computation experiments for translation to learning activities geared towards their classrooms. Integrated discussion and observation activities will bridge the participants' experiences to the social and ethical implications of the technology and the way the technology is deployed within public and industry laboratories.

Progress 09/01/16 to 08/31/17

Outputs
Target Audience:This project targets Graduate students, undergraduate students, postdoctoral researchers, and scientific staff involved in this project through research and laboratory experiences. Students at all levels via classroom teaching and instruction as well as formal and informal scientific and professional exchange. Colleagues in the plant research community and other life science areas through the Crop Bioengineering Consortium website, peer-reviewed publications, conference presentations, and formal or informal scientific and professional exchange. Regulatory and governance experts through invited lectures and symposia K-14 teachers through summer training workshops. The general public through news releases, invited lectures, guided tours of the ISU Center for Plant Transformation and via website information. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project involved one PhD student and one postdoc research associates (80%). Also involved are three professional staff in gRNA designing and analysis, DNA construct building, genetic transformation of corn and rice, and transgenic plant analysis. How have the results been disseminated to communities of interest?This project is to improve an open-sourced CRISPR guides design tool CGAT developed by Carolyn Lawrence-Dill at Iowa State University, who is one of the co-PDs of this project. Any data and results generated from this work will be disseminated to plant scientists via conference and symposium presentation and peer-reviewed journal publications; and to biology and science major undergraduate students via classroom lectures. Most significantly, CGAT will be improved so this tool can be used by the community for more effective gRNA design. In addition, the improved CRISPR vectors and genome-editing procedures will be distributed to the community via ISU Plant Transformation Facility directed by PD Wang. Non-technical summary of the technology development and improvement are being disseminated to legislators and regulatory officers via workshop and guided discussions; and to general audience via guided tours and hands-on workshops during summer. What do you plan to do during the next reporting period to accomplish the goals?Specific actions to take in the next reporting period to achieve the proposed objectives: Objective 1. Continue to analyze and improve predictions made by CGAT open source CRISPR gRNA design and off-target prediction tool. Objective 2. Continue to validate off-target cleavage and reduction by high-fidelity nucleases using an in vitro approach. Objective 3. Continue to validate off-target activity on transgenic events in planta. Molecular analysis of transgenic maize and rice plants will be carried out. On-target and off-target studies will be performed. Objective 4. Continue to Develop and carry out outreach and communication activities describing CRISPR technology and agriculture biotechnology. They will be in forms of lecture, guided discussion, hands-on practice, or the combination of all activities.

Impacts
What was accomplished under these goals? Overall impact statement: Issue: The most advanced genome-editing technology CRISPR/Cas9 (clustered regularly-interspaced short palindromic repeats with associated Cas9 protein) system can be used to generate site-specific mutations for trait improvement in many plant species. However, it is not clear how frequent the unexpected and unwanted DNA changes occur at non-target areas of genomes (off-target effects) in these edited plants. How we addressed the issue & results: This research aims at developing and improving a CRISPR design tool that can reduce mutation frequencies on unintended DNA sequences (off-targets). The data generated from three specific research objectives can be used to improve the predictive reliability of plant CRISPR specificity through the improvement of an open-source CRISPR design tool developed by Iowa State University. In addition, the project develops and carries out an outreach program for research and general communities. Impact: This research is directed towards the development of a bioinformatics screening tool for unbiased prediction of CRISPR reagent design and regulatory assessment. The development of validated tools to accomplish improved design and prediction of genome editing outcomes is a well-recognized need for safety assessment, which has not yet been addressed for plant genome editing. Such a tool, when used as a conservative screen, can help to identify, on a case-by-case basis, the degree to which further follow-up studies are needed to address downstream effects in genetically engineered (GE) organisms. This proposal addresses BRAG standard program area 5(d) "Research addressing off-target genotypic and/or phenotypic effects in GE organisms developed using genome editing technology." Objective 1. Analyze and improve predictions made by a CRISPR design tool developed by Iowa State University with the goal to reduce off-target mutations (Year 1 of 3; 30% completed). 1) Major activities completed / experiments conducted: Two target genes, one for maize and one for rice, were chosen based on their known phenotypes. The genes are glossy2 (gl2; related to leaf-gloss) in maize and phytoene dehydrogenase (pds1; related to albinism) in rice. We used several online CRISPR tools to identify potential CRISPR guides which target these genes of interest. The tools used included three public on-line softwares CGAT (designed by Iowa State Univ), CHOP-CHOP, and CRISPR-P. We cross-referenced the identified guides from the software tools and created a list of favorable CRISPR guides identified and highly-ranked by more than one tool. 2) Data collected: The gRNAs identified in the above process were synthesized and cloned into CRISPR vectors that carry a Cas9 gene cassette. The binary vectors designed for maize and rice transformation were built. 3) Summary statistics and discussion of results: Will be available next reporting period. 4) Key outcomes or other accomplishments realized: gRNAs design to target two maize and rice genes were performed extensively to compare outcomes obtained from different online gRNA design softwares. Off-target prediction based on reference genome analysis was also done. Objective 2. Validate off-target cleavage and reduction by high-fidelity nucleases using an in vitro approach (Year 1 of 3; 30% completed). 1) Major activities completed / experiments conducted: Validation experiments have been performed for wild-type (WT) Cas9. In parallel with these experiments, we have begun efforts to clone and purify high-fidelity versions of Cas9 (HF-Cas9). Multiple HF-Cas9 constructs have been cloned, and expression and purification protocols are currently being optimized. 2) Data collected: sgRNA and target plasmids were generated for the pds1 gene exon 1. In vitro cleavage assays were carried out using reconstituted SpCas9-sgRNA complex. Cleavage was observed for both the 18 and 20 base on-target plasmids, but not observed for the off-target plasmids. 3) Summary statistics and discussion of results: Our initial results indicate that the preliminary targets chosen for study will have very low or undetectable off-target effects at the predicted sites in in planta studies. 4) Key outcomes or other accomplishments realized: Our studies reveal that cleavage is completely blocked for off-target sites predicted for the pds1 target. These results suggest that off-target effects will not be observed in planta experiments for these targets. In the second stage of this study, we will select targets with more closely related off-target sites. This will be necessary to observe off-target cleavage by WT Cas9, and to determine the improvement of off-target effects with HF-Cas9. Objective 3. Validate off-target activity on transgenic events in planta (Year 1 of 3; 30% completed). 1) Major activities completed / experiments conducted: Two target genes, one for corn (gl2, a gene responsible for the synthesis and deposition of epidermal wax layer in young leaves) and one for rice (pds, a gene responsible for carotenoid biosynthesis), were chosen based on their known phenotypes. Two target genes, gl2 gene for maize and pds gene for rice, were chosen based on their known phenotypes. gRNAs designed in Objective 1 were cloned into binary vectors for Agrobacterium-mediated transformation. Maize and rice transformation have been initiated and ongoing. 2) Data collected: Transgenic maize and rice events are emerging and being analyzed. To date, a total of 102 independent maize plants representing 12 transgenic event and 544 rice plants representing 136 events have been produced. 3) Summary statistics and discussion of results: Analysis of transgenic maize and rice plants for on-site and off-site mutations is on-going and the results will be available in next reporting period. 4) Key outcomes or other accomplishments realized: Transgenic maize and rice plants carrying CRISPR reagents targeting to specific genes are being produced. Objective 4. Develop and carry out outreach and communication activities describing CRISPR technology and its robustness for targeted editing to both the research and regulatory communities (Year 1 of 3; continuous effort). Apr13, 2017, Co-PD Wolt organized a half-day workshop on modern breeding and genome editing technology for breeders. May 10, 2017, Co-PD Wolt provided ½-day training for Ghanaian regulators in risk communication for genetic engineering. University of Maryland, Eastern Shore, Cochran Fellowship Program, USDA-FAS. Jun6, 2017, Co-PD Wolt presented perspectives on environmental risk assessment (ERA) at the 14th International Symposium for Biosafety of Genetically Modified Organisms, Mexico Jun28, 2017, PD Wang conducted a half-day hands-on workshop for a group of high school science teachers on CRISPR genome-editing and genetic transformation. This was part of Iowa State University Biotechnology summer training activity for Iowa high school teachers. Jul9, 2017, Co-PD Wolt gave presentation to the Agriculture and Natural Resource Committee at the Midwestern Legislative Conference (MCL) of the Council of State Governments (CSG) Jul17, 2017, PD Wang gave dinner speech to Iowa STEM Professional Development Workshop Jul17, 2017, Co-PD Wolt gave ½-day training for Philippine regulators in risk communication for genetic engineering. University of Maryland, Eastern Shore, Cochran Fellowship Program, USDA-FAS. Jul20, 2017, Co-PD Wolt gave presentations on biosafety regulation and regulatory aspects of genome-edited crops to representatives of the Korean Biotech Safety/Risk Evaluation Committee. Ames, US Grains Council. Aug16, 2017, Co-PD Wolt and PI Wang gave lectures to a group of visiting scientists from Jiling Agriculture Science Academy, China. Integrated discussion and hands-on activities bridge the participants' experiences to the social and ethical implications of the technology and the way the technology is deployed within public and industry laboratories.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Wolt, JD, Wang, K, Sashital, D, Lawrence-Dill, CJ. Achieving Plant CRISPR Targeting that Limits Off-Target Effects. Plant Genome, Volume 9. doi: 10.3835/plantgenome2016.05.0047. (2016)
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Wolt, J, Yang, B, Wang, K, Spalding, M. Regulatory aspects of genome-edited crops. In Vitro Cellular & Developmental Biology  Plant. DOI 10.1007/s11627-016-9784-3 (2016)
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Wolt, JD, Wang, K, Yang, B. The regulatory status of genome-edited crops. Plant Biotechnology J. 14: 510-518 (2016).
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Wolt JD. 2017. Safety, security, and policy considerations for plant genome editing, pp. 215-241, In Donald P. Weeks and Bing Yang, editors: Gene Editing in Plants, Vol 149, PMBTS, UK: Academic Press.