Massively Multiplexed Zinc Finger Protein Engineering

Zinc fingers are specialized proteins that bind to DNA. Due to their ability to target highly specific DNA sequences, zinc fingers offer great potential for gene therapy and personalized medicine: recently, they were shown to be effective in conferring HIV resistance and treating hemophilia in mice. In the past, however, designing new zinc fingers - a necessity for individualized gene therapy - has been prohibitively expensive and time consuming. For more information about zinc fingers, see here.

For our project, we created and tested thousands of zinc fingers at a cost feasible for most labs. To do so, we harnessed three novel synthetic biology technologies: chip-based synthesis^[1], which allows for thousands (even millions) of DNA strands to be synthesized concurrently, multiplex automated genome engineering (MAGE)^[2][3] and lambda red recombineering ^[4][5][6], which both make possible direct edits of the genome of organisms, rather than using small, cumbersome plasmids.

To do this, our project has three main steps:

1. Design

Use a bioinformatics approach to predict 55,000 zinc finger sequences.

As the structure and binding interactions of zinc fingers are not yet understood, our project utilizes bioinformatics and computational analysis of the limited existing data to make “educated guesses” of what amino acid sequences will bind to our desired target sequence.

Team members chose 6 target sequences in the human genome (finding these sequences using a binding site finder tool written by a team member) that no currently-existing zinc finger is able to bind to: genes that cause colorblindness, some types of cancer, and high cholesterol. These targets were chosen after conducting an extensive literature search, with the most useful information coming from a previous zinc finger study called CODA^[7].

Team members emailed the OPEN consortium^[8] and Dr. Anton Persikov^[9] to acquire their respective databases of zinc fingers: Persikov had compiled the results of the past 20 years of zinc finger research (everything before OPEN), and OPEN had found many more since then. In all, around 1,700 unique DNA triplet/zinc finger helix pairs were discovered. Team members then decided how to analyze this data for the best chance of binding our target sequences, and programmed their ideas into a Python program to generate 55,000 potential zinc fingers.

For a complete overview of how our zinc fingers were designed, see Design.

2. Synthesize

Use chip-based DNA synthesis to make 55,000 sequences simultaneously, then insert the oligos into E.coli.

Once our team generated these 55,000 sequences, we needed a way to bring them from a computer's memory into the real world. Using chip synthesis (generously contributed by Agilent Technologies - a sponsor of iGEM - in partnership with our mentors the Church Lab), all of these sequences were built simultaneously, then sent to us in a single 50μL tube.

For more information about how the chip was created, see the original [http://www.nature.com/nbt/journal/v28/n12/full/nbt.1716.html 2010 paper by Kosuri et al].

Chip synthesis results in a pool of single stranded oligos, designed with primer tags, which allow for the amplification of specific sub-pools: in our team's case, we used 6 sub-pools, one for each of our target sequences.

For a complete overview of how our zinc finger DNA went from chip to being expressed in E.coli, see Synthesize and Results of Synthesize.

3. Test

Use a genomic metabolic selection system to test which zinc finger sequences successfully bind DNA.

After creating and expressing 55,000 novel zinc finger sequences, our team needed to determine which ones effectively bind to their respective target sites. By tying zinc finger binding to cell survival (using an efficient selection system) all cells without successful binders would die, and thus living colonies indicate a viable zinc finger.

Team members constructed a one-hybrid selection system based off a metabolic system designed by Meng et al^[10] which tied zinc finger binding to histidine production. When grown in media without histidine, the cells can only survive if a zinc finger-omega subunit of RNA polymerase (also knocked out in the strain) fusion protein binds successfully and initiates creation of histidine.

Where our team's selection departs from the one described by Meng and others is its use of a genome-based rather than plasmid-based system. Not only did team members knock out HisB, PyrF, and rpoZ ourselves using the newly developed techniques of MAGE and lambda red, we also inserted the zinc finger binding site construct directly into the genome instead of expressing it in the cell on a vector.

For a complete overview of how our selection system was designed and tested, see Test and Results of Selection.

Thus, our zinc fingers and their clinical applications are a new technology that maximize efficiency and decrease cost: we anticipate that future iGEM teams will find great use for chip-based synthesis, MAGE, and lambda red.

See here for our abstract and detailed project description.

Technological Applications

The novel methods we employed in our project have the potential to revolutionize synthetic biology practices, and the way that future iGEM competitions are conducted. To learn more about the technological applications of our project, please see our Technology page.

Zinc Finger Background

What are Zinc Finger Proteins (ZFPs)?

Function

ZFPs are found commonly in nature as a class of special transcription factors that bind to DNA, thus regulating gene expression. Zinc finger function was first studied using zinc finger protein Zif268.

Structure

ZFPs consist of smaller subunits called "fingers" which each contain a zinc finger binding helix that binds to unique DNA sequences. These fingers are linear and linked together by the "zinc finger backbone", a series of approximately 21 amino acids.

• Cis2His2 ZFPs have three main structural components:
  • Zinc finger binding helix
  • Linker region
  • Zinc ion that is coordinated by two cysteine residues and two histidine residues.

Helpful Zinc Finger Links

[http://en.wikipedia.org/wiki/Zinc_finger Zinc Fingers on Wikipedia]

• A more detailed introduction to zinc fingers.

[http://compbio.cs.princeton.edu/zf/ Predicting DNA Recognition by C2H2 Zinc Finger Proteins]

• A program useful for predicting how well a given amino acid sequence will bind to a given DNA sequence

[http://www.zincfingers.org/default2.htm The Zinc Finger Consortium]

• Information & helpful resources for zinc fingers

[http://www.jounglab.org/ Joung Lab]

• Information about Dr. Joung's extensive work with zinc fingers

References

1. Sriram Kosuri, Nikolai Eroshenko, Emily M LeProust, Michael Super, Jeffrey Way, Jin Billy Li, George M Church. (2010). Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nature Biotechnology, 28(12):1295-9. [http://www.nature.com/nbt/journal/v28/n12/full/nbt.1716.html]

2. Harris H. Wang, Farren J. Isaacs, Peter A. Carr, Zachary Z. Sun, George Xu, Craig R. Forest, George M. Church. Programming cells by multiplex genome engineering and accelerated evolution. (2009). Nature, 460(7257):894-8. [http://www.nature.com/nature/journal/v460/n7257/full/nature08187.html]

3. Isaacs FJ, Carr PA, Wang HH, Lajoie MJ, Sterling B, Kraal L, Tolonen AC, Gianoulis TA, Goodman DB, Reppas NB, Emig CJ, Bang D, Hwang SJ, Jewett MC, Jacobson JM, Church GM. (2011). Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science, 333(6040):348-53. [http://www.sciencemag.org/content/333/6040/348.full]

4. Yu, D., H. M. Ellis, et al. (2000). An efficient recombination system for chromosome engineering in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 97(11): 5978-5983.[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC165854/]

5. Mosberg JA, Lajoie MJ, Church GM. Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. Genetics 2010;186:791-799.[http://www.genetics.org/content/186/3/791]

6.(Supporting material for) Isaacs FJ, Carr PA, Wang HH, Lajoie MJ, Sterling B, Kraal L, Tolonen AC, Gianoulis TA, Goodman DB, Reppas NB, Emig CJ, Bang D, Hwang SJ, Jewett MC, Jacobson JM, Church GM. (2011). Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science, 333(6040):348-53.[http://www.sciencemag.org/content/suppl/2011/07/13/333.6040.348.DC1/Isaacs.SOM.pdf]

7. Jeffry D Sander, Elizabeth J Dahlborg, Mathew J Goodwin, Lindsay Cade, Feng Zhang, Daniel Cifuentes, Shaun J Curtin, Jessica S Blackburn, Stacey Thibodeau-Beganny, Yiping Qi, Christopher J Pierick, Ellen Hoffman, Morgan L Maeder, Cyd Khayter, Deepak Reyon, Drena Dobbs, David M Langenau, Robert M Stupar, Antonio J Giraldez, Daniel F Voytas, Randall T Peterson,Jing-Ruey J Yeh, J Keith Joung. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA)(2011). Nature Methods 8, 67–69. [http://www.nature.com/nmeth/journal/v8/n1/full/nmeth.1542.html]

8. Morgan L. Maeder, Stacey Thibodeau-Beganny, Anna Osiak, David A. Wright, Reshma M. Anthony, Magdalena Eichtinger, Tao Jiang, Jonathan E. Foley, Ronnie J. Winfrey, Jeffrey A. Townsend, Erica Unger-Wallace, Jeffry D. Sander, Felix Müller-Lerch, Fengli Fu, Joseph Pearlberg, Carl Göbel, Justin P. Dassie, Shondra M. Pruett-Miller, Matthew H. Porteus, Dennis C. Sgroi, A. John Iafrate, Drena Dobbs, Paul B. McCray Jr., Toni Cathomen, Daniel F. Voytas, J. Keith Joung. Rapid “Open-Source” Engineering of Customized Zinc-Finger Nucleases for Highly Efficient Gene Modification (2008). Molecular Cell Volume 31, Issue 2, 25 July 2008, Pages 294-301.[http://www.sciencedirect.com/science/article/pii/S1097276508004619]

9. Anton Persikov, PhD. [http://www.princeton.edu/~persikov/publications.html]

10. Xiangdong Meng, Michael H Brodsky, Scot A Wolfe. A bacterial one-hybrid system for determining the DNA-binding specificity of transcription factors. (2005). Nature Biotechnology, 23(8): 988-994. [http://www.nature.com/nbt/journal/v23/n8/pdf/nbt1120.pdf]