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AbstractTranscription activator-like effector nucleases (TALENs) are created by joining a TAL effector DNA binding domain to a DNA cleavage domain1,2. We intend to produce six TALENs as a proof of concept to modify the genome of Glycine max (soybean). This research project was designed to test the construction accuracy and efficiency of these six TALENs prior to their introduction into G. max.

Figure 1: Sample TALEN Design

Challenge:1. Each TALEN target must begin with a T.2. Spacer between TALENs must correspond to one of the

tested backbone vectors.3. No long stretches of A’s and T’s.4. Cut two or more long form genes with one TALEN pair to

make knock outs.5. Do not cut short form genes.6. The TALEN must match as many nucleotides as possible

- especially near the 5’ end of each target.

Outcome:1. Can target the two most closely related long form genes.2. Two mismatches at the 8th and 16th positions of TALEN 1 -

could test the NN, NS or N* RDVs at this position using the yeast assay.

3. TALEN 2 is an exact match for both long form genes.4. Should be able to cut either or both genes and truncate in

exon 5.5. TALEN 1 has multiple mismatches with the short forms

and may not bind.6. TALEN 2 has multiple mismatches with the short forms,

which includes the required 5’ T so this should not bind and no cutting is expected.

Acknowledgments I would like to thank David Wright and Martin Spalding for helping me understand the project and allowing me to use their facilities to conduct this research. I would also like to thank Adah Lesham for coordinating the RET program which provided this opportunity to work in the lab. My research was funded by the National Science Foundation, grant number NSF DBI-0638418 (PI Dr. Jonathan Wendel). Finally, I would like to thank Iowa State University for the use of their facilities.

Conclusions• Successfully completed and tested TALEN pair for

Exon 3

• Successfully completed and tested TALEN pair for Exon 5

• Completed and tested left TALEN for Exon 7 with remaining right TALEN yet to be tested

• These TALENs will be tested against the target areas from the short forms of the gene to verify they do not cut the short forms

• When all testing is finished, these TALENs will be transferred into Glycine max (soybean) and tested for efficacy

Marc Benedict1, David Wright2, and Martin Spalding21Graettinger-Terril High School, Graettinger, Iowa; 2Department of Genetics, Development and Cell Biology, Iowa State

University, Ames, Iowa

Literature cited

1 Boch, Jens et al. "Breaking the code of DNA binding specificity of TAL-type III effectors." Science 326.5959 (2009): 1509-1512.

2 Moscou, Matthew J, and Adam J Bogdanove. "A simple cipher governs DNA recognition by TAL effectors." Science 326.5959 (2009): 1501-1501.

3 Li, Ting, and Bing Yang. "TAL Effector Nuclease (TALEN) Engineering." Enzyme Engineering (2013): 63-72.

Engineering TAL Nucleases (TALENs) for Use in Soybeans

Materials and MethodsThe TALENs were first constructed in E. coli by ligating together short sets of repeat variable diresidues (RVDs) that each carry part of the desired sequence specificity then these sets are ligated together in a yeast expression vector to form the final TALEN3. The targets were assembled by cloning primer pairs that are representative of the target and inserted into a reporter yeast plasmid (LacZ). After confirming the presence of intact TALENs and their respective targets in the yeast vectors, these plasmids were introduced into haploid yeast strains YPH 499 (TALEN) and YPH 500 (LacZ-Target). The strains of yeast containing the TALEN and its target were mated. The resulting diploid yeasts were tested using an x-gal/LacZ assay to confirm TALEN functionality.

Figure 2. Yeast Recombination Assay

A. B.

1. The target sequence is cloned between a duplicated region LacZ.

2. Target sequence is cleaved by TALENs.

3. Recombination between the duplicated sequence results in a functional gene.

A. The yeast recombination assay uses the correction of a dysfunctional gene as a test for TALEN functionality.

B. pDW3054 is a mating type (a) that expresses the TALEN. pDW3036 is a mating type (α) expresses the target sequence. When a fuses with α through mating, the TALEN and the target can interact. If the TALEN is functional, then the target site is cleaved, and LacZ is reconstituted by recombination resulting in a blue color. Functionality is estimated based on time to achieve 100% blue colonies.

Lac acZ

LacZ

Target Sequence

Lac acZ

Results A

B

C D E

F G

Figure 3: Yeast Assay for Long Form Exon 5 TALEN Pair

(NOTE: Each TALEN appears in both a high copy and low copy plasmid. Left TALEN has two target variants.)

A. Positive Control (known TALEN against known target)

B. Negative Controls: Same TALEN against both left targets and right target

C. Left TALEN against first left target

D. Left TALEN against second left target

E. Left TALEN against right target

F. Right TALEN against both left targets

G. Right TALEN against right target