The goal of Kiran Musunuru's lab in Harvard's Department of Stem Cell and Regenerative Biology is to understand the basis for cardiovascular and metabolic human diseases. They do that by studying patients to uncover new gene variants associated with conditions of interest, then studying those variants in model systems: either human cells or mice.
In a recent issue of Cell Stem Cell, Musunuru, Chad Cowan and their colleagues describe a much more efficient tool for doing that disease modeling work in human pluripotent stem cells: a transcription activator-like effector nuclease (TALEN) kit consisting of 834 plasmids. The researchers showed they could use their TALEN kit to quickly and efficiently generate human stem cells edited to carry mutant versions of 15 different disease-associated genes.
Addgene spoke to Musunuru about how the new kit works, the research the kit now makes possible, and how it compares to his CRISPR/Cas9 system.
Addgene: For people unfamiliar with this new TALEN kit, can you describe it for us?
Musunuru: TALENs are a type of genome editing tool with which one can introduce changes into the genomes of mammalian or other cells. TALENs are engineered proteins that comprise a DNA binding domain and an endonuclease domain. What’s distinctive is that the DNA-binding domain is modifiable and can be programmed to bind to essentially any DNA sequence desired. This technology allows you to create a pair of designer proteins that will introduce a double-strand break in the genome anywhere you would like. Cells repair those breaks in one of two ways: by putting the two ends back together or using a homologous template provided by the sister chromosome. As cells take those two ends and put them back together, there are occasional errors. That can be advantageous if you are trying to knock out a gene. One can also exploit homology-directed repair by flooding the cell with a mutant template and fooling the cell into using that template for repair. By using these two scenarios, you can either knock out a gene or knock in a mutation.
Addgene: How do you use this kit to target a particular DNA sequence?
Musunuru: The kit includes 832 plasmids containing prefabricated pieces that allow you to build a DNA-binding domain to recognize any desired 15 base pair sequence in the genome in rapid fashion. The two remaining plasmids in the kit have the rest of the protein in place including FokI endonuclease domain as well as fluorescent proteins used to sort the cells after the plasmids have been introduced by transfection or electroporation. All you do to create a protein that binds those 15 base pairs is take four plasmids out of the library in the appropriate combination and do a restriction enzyme digest to cut out the inserts followed by a five-piece ligation with the four inserts and the master plasmid in one step. The kit also has other unique aspects that make it especially well-suited for use in stem cells. (See the complete protocol at StemBook).
Addgene: How are you now using this tool?
Musunuru: We have been using the kit to target a large number of genes involved in cardiovascular and metabolic diseases. We are knocking out genes that have been implicated in disease or, in cases where particular variants or mutations have been reported in people, we can use this kit to knock in those mutations. Once that's done in stem cells, we can differentiate those mutant and wild type stem cells into any cell type relevant to the disease in question – cardiomyocytes, adipocytes or neurons, for instance – to see how those mutations influence the disease process. In the Cell Stem Cell paper, for example, we generated hepatocytes to examine the effect of a gene on cholesterol metabolism. The TALEN kit is quite useful in generating the mutant cell lines that one needs to do disease modeling in stem cells.
Addgene: Tell me about the history. How had investigators done this kind of work before?
Musunuru: In the "olden days," not even five years ago, scientists relied on traditional homologous recombination. That technology had been used for decades to generate knock-out mice, but for various reasons it is not nearly as efficient in human stem cells. What genome editing tools allow you to do is greatly increase the efficiency of the genome editing process. The first really popular tool was the zinc finger nucleases. Unfortunately, it is time consuming and difficult for investigators without a lot of expertise to create zinc finger nucleases that will bind desired sequences. TALENs are much more reliable. If you design them for a particular sequence, there is a much better chance they will bind that sequence. That’s the revolutionary aspect: TALENs take a technology that was difficult for all but the most experienced users and puts it in the hands of pretty much all investigators.
Our kit makes it extremely simple to design TALENs. It only takes two to three days from the moment you are designing a TALEN on paper to a finished plasmid. With our protocol, it is feasible to produce genetically modified cells in less than a month's time.
Addgene: What are the implications of this kit in terms of understanding human disease?
Musunuru: The power here is that you could potentially sequence a large number of individuals and find several mutations in the same gene or in multiple genes, all of which seem to contribute to a particular disease state. You could use the kit to insert all of those variants into stem cells with the same genetic background and do an apples-to-apples comparison of all those mutations. If you make induced pluripotent stem (iPS) cells from patients and try to compare those lines, they may all have the mutations but they will also have different genetic backgrounds. It becomes more of an apples-to-oranges comparison.
Addgene: Can you compare the TALEN kit to CRISPR/Cas9? When would you choose one or the other?
Musunuru: The CRISPR/Cas9 system is easier to use as testified by the fact that the TALEN kit includes 834 plasmids for the pieces you need to assemble. Our system for CRISPR/Cas9 (also available from Addgene) only has two plasmids. In many cases, CRISPR may be more efficient. You might often start with CRISPR/Cas9, but TALENs do have more flexibility in terms of the target site. Also, any time you create double-strand breaks, there is a possibility of introducing a mutation somewhere else in the genome. It remains to be seen which system is cleaner in terms of minimizing off-target effects, but the data to date suggest TALENs may be cleaner than CRISPR/Cas9.
Quirong Ding et al. "A TALEN genome-editing system for generating human stem cell-based disease models." Cell Stem Cell. 12, 238-251 (7 February 2013).