TOWARDS GENE STACKING METHOD FOR CROP IMPROVEMENT: FEASIBILITY ANALYSIS FOR GENE STACKING IN PLANT GENOMES BY STACKING MULTIPLE GENES IN RICE GENOME AND TESTING THEIR STABILITY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1001205
• Project Start Date: Oct 1, 2013
• Project End Date: Sep 30, 2016
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703

Performing Department
Crop, Soil & Environmental Sciences

Non Technical Summary
Developing transgenic crops with multiple genes is important for effective weed and pest management. Traditional transformation methods are generally impractical for multigene introduction because these methods often introduce more than one copy (or truncated copies) of genes into random sites, leading to silencing of one or more genes. New methods that can precisely add foreign genes into a chosen site can simplify multigene transformation. This approach is referred to as "molecular stacking", and can be achieved either by inserting a single DNA fragment containing multiple genes or inserting genes one by one. There are several outstanding questions, such as, what is the stability if the multigene locus and which molecular tools would be efficient in developing a gene stacking approach. This project will address these questions, which are important for developing the method of gene stacking.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1530	1040	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1530 - Rice;

Field Of Science
1040 - Molecular biology;

Keywords

gene stacking

multigene transformation

dna recombination

endonucleases

transgenic plants

Goals / Objectives
The long term goal of this project is to develop a method of iterative plant transformation suitable for stacking multiple genes into a single genomic site. However, first it should be tested whether stacking of multiple genes into a single genomic site is equivalent to single gene transformation, a commonly used method. So far, only a limited data is available on the stability of genes within the "molecular stack". Towards this goal, experiments will be conducted to test the feasibility of molecular tools and efficacy of the multigene locus. The results of these experiments will help in (a) testing the suitabilityof gene stacking in GM crops, (b) determining the stability of genes within the stacks, and (c) developing gene stacking methods.

Project Methods
Rice variety Nipponbare or Taipei 309 will be used for all tissue culture and transformation experiments. Standard tissue culture and transformation procedures will be used. The embryogenic callus derived from mature seeds will be used for genetic transformation by particle bombardment. DNA constructs containing multigene cassette will be developed by standard cloning techniques. Each gene will be driven by one of the three constitutive promoters (35S, Ubi-1, Act-1) and terminated by nos 3' terminator. Thus, there will be repetitive elements in the construct, and the project will be able to address the stability of clones and genetic locus containing DNA repeats. Use of repetitive elements is deliberate in this project because in future many repetitive elements are likely to be used in GM crops. Multigene cassette insertion will be tested in a founder line, T5, developed earlier using particle bombardment method. Transgenic clones will be isolated through selection on bialaphos (5 mg/L) and analyzed by (a) Southern hybridization to determine structure of the locus, (b) real time PCR on total RNA to determine expression of each gene. In the second part of the project, the efficiency of I-SceI and I-CeuI will be tested in rice genome. First a DNA construct containing I-SceI or I-CeuI recognition sites will be developed, and introduced into rice genome. Then I-SceI or I-CeuI expression cassette will be introduced into the selected clones. The doubly-transformed cell lines will be analyzed for the DSB induction and repair of the locus by isolating PCR fragments and sequencing them. The clones showing I-SceI/I-CeuI activity will be regenerated to assess homogeneity of I-SceI/I-CeuI reaction and inheritance of the repaired locus. For this, primary transgenic lines (T0) will be analyzed by Southern hybridization to determine the structure of the I-SceI/I-CeuI site locus, and T1 progeny for the inheritance of the repaired locus.

Progress 10/01/13 to 09/30/16

Outputs
Target Audience:Research communities at academic, industry and government institutions were reached by this project's efforts during this reporting period. The progress in the project was presented at regional and national meetings by students as described in the publications section of this report. Changes/Problems:Due to the observed toxicity of nucleases in transgenic plants, genetic crosses could not be done. Therefore, tissue culture of target lines will be subjected to particle bombardment to generate transient expression of nucleases for evaluating their effciency on chromosomal fragment deletion. We have also started testing the effciency of CRISPR-Cas9 in targeted gene deletions in rice. Transgenic rice plants containing CRISPR constructs were generated, which showed targeted excision of GUS gene. These plants will be used for determining heritability of the excision locus. What opportunities for training and professional development has the project provided?The project involved 1 graduate student and 1 undergraduate student. These studentsdeveloped DNA constructs, transgenic plants and carried out molecular analysis for detection of genomic excisions. How have the results been disseminated to communities of interest?The graduate students presented a poster for 3 different conferences in 2016 and an abstract of each presentation was published in the conference proceedings (see the publication section in this report). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The approach of gene stacking involves the use of recombinases and nucleases. The effciency and efficacy of recombinases is already known in the model plant, rice. Therefore the goal of this project is to test the efficacy of nucleases. We selected I-SceI and CCR5 ZFN based on prior reports of their use in plants. Towards this we developed gene constructs both overexpressing these nucleases and inducible expression, and generated transgenic rice plants. We found that overexpression of both I-SceI and ZFN was possibly toxic to rice plants; transgenic plants with induced expression were recovered. However, low seed set and poor pollen viability indicated toxicity of nuclease in these plants. These observations suggest that genetic crosses to introduce nuclease expression might not be a viable approach. Instead, we also generated CCR5 and I-SceI target lines by transforming constructs bearing target sites into rice. These lines will be used for transient expression of nucleases to direct excision.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Srivastava V, Underwood J, and Zhao S. Dual-targeting by CRISPR/Cas9 for precise excision of transgenes from plant genomes. J. Plant Biotech. DOI : 10.1007/s11240-016-1166-3
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Pathak B, Underwood J, Nandy S, Zhao S, Srivastava V (2016) A streamlined approach of gene stacking based on site-specific recombinases and nucleases. Society of In Vitro Biology, Jun 11-15, 2016, San Diego, CA.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Pathak B, Nandy S, Zhao S, Underwood J, Srivastava V (2016) I-SceI mediated marker gene deletion from the rice genome. Annual Meeting of Plant Imaging Consortium, Fayetteville, AR, July 7  8, 2016.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Pruett E, Nandy S, and Srivastava V (2016) Towards trait stacking in crops: Efficiency of I?SceI nuclease in excising DNA fragments from Arabidopsis genome. American Society of Plant Biologist  Southern Section. April 2  4, 2016. Denton, TX.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:This project provided research experience to 1 undergraduate student, who has worked for 2 semesters. The student is learning basic molecular biology techniques and helping us analyze the transgenic clones developed in this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project trained a postdoctoral fellow in research project management and data presentation. In addition, a graduate student and an undergraduate student started working in this project, receiving training in plant biotechnology. How have the results been disseminated to communities of interest?The results of this project were presented in the annual meeting of the Society of In Vitro Biology at Tuczon, AZ, May 31 - Jun 4, 2015. The first presentation below was an oral presentation, and the second is a poster presentation by the undergraduate student (E. Pruett). (a) Srivastava V, Nandy S, and Zhao S. Iterative modification of transgene locus towards multigene stacking into a single genomic site. In Vitro Biology, Volume 51, Number 4, P-1023. (b) Pruett E, Underwood J, Srivastava V. Evaluation of lox-flanked pollen-specific gene excision from Arabidopsis thaliana and Nicotiana tabacum genomes by Cre activity. In Vitro Biology, Volume 51, Number 4, P-2027. What do you plan to do during the next reporting period to accomplish the goals?We plan on the following activities in the next year: (a) determine the activities of heat-shock nuclease gene in marker excision from the site-specific integration locus (b) characterize the new target lines for its effciency in gene stacking by Cre-lox (c) determine the effciency of CRISPR in marker gene excision.

Impacts
What was accomplished under these goals? Proof-of-concept experiments were completed using rice tissue culture and transformation for (a) recombinase-mediated site-specific gene integration, and (b) nuclease-mediated marker gene excision towards building a marker-free multigene stacked locus. We used Cre-lox for inserting genes into the pre-integration site, and I-SceI and CCR5-ZFN for excising marker genes. The plant lines were analyzed by PCR and Southerns to validate the stacked locus and structure before and after marker-excision. We found that site-specific gene integration efficiency are determined by the extraordinary recombination efficiencies of Cre-lox and FLP-FRT systems delivering 20 - 50% recovery of site-specific integration lines. Then we found that I-SceI gives ~30% efficiency in excising marker gene from the integration locus without creating large insertions-deletions. The integrated genes, GFP and GUS are expressed ain all stacked lines strongly. We did not quantify gene expression or GFP protein/GUS activity as all lines showed strong activities. We also developed new rice target lines for gene stacking experiments, which were analyzed by Southerns to determine the construct copy number. Currently, we have two new single-copy target lines that are in greenhouse awaiting seed set.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: " Nandy S, Zhao S, Pathak BP, Manoharan M, Srivastava V (2015) Gene stacking in plant cell using recombinases for gene integration and nucleases for marker gene deletion. BMC Biotechnol. 15:93. doi: 10.1186/s12896-015-0212-2.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Srivastava V, Thomson J (2015) Gene stacking by recombinases. Plant Biotechnol J. doi: 10.1111/pbi.12459.
• Type: Book Chapters Status: Published Year Published: 2015 Citation: Srivastava V and Ow DW (2015) Simplifying transgene locus structure through Cre-lox recombination. Plant Gene Silencing, Methods in Molecular Biology 1287 (eds. K. S. Mysore and M. Senthil-Kumar), Springer Protocols, pp 95 - 103.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: This project provided research experience to 3 undergraduate students, one whom has worked for 2 semesters. The student is learning basic molecular biology techniques and helping us analyze the transgenic clones developed in this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? We will develop new founder lines for stacking C4 genes in rice genome. For this, a new DNA construct is under preparation, which will be used for rice transformations. We will also test whether a new nuclease, ZFN, can be used for marker excision as effectively as I-SceI.

Impacts
What was accomplished under these goals? Proof of concept was developed for iterative gene integration and marker excision that are key steps of the proposed gene stacking strategy. using an existing founder line of rice in Cv Nipponbare background, we integrated GFP gene into the selected genomic site in the rice genome by Cre-lox recombination. We then excised the marker gene (Bar gene) by I-SceI enzyme activity, Next, we inserted GUS gene into the site using FLP-FRT recombination system. These experiments showed that a combination of site-specific recombination systems such as Cre-lox and FLP-FRT and rare nucleases such as I-SceI could be used for developing iterative transformation protocol. This protocol will allow stacking multiple genes into a single genomic site, simplifying multi-trait breeding.

Publications