Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Not long ago, the ability to effect permanent, targeted genomic changes in mammalian cells relied on laborious, lengthy and somewhat random techniques. The advent of the zinc finger nuclease (ZFN) system—a DNA-recognition domain fused to the obligate-dimer FokI endonuclease—enabled researchers to design reagents that bind to and cut a stretch of chromosome of their own choosing [1]. After the cutting, the cell’s endogenous mechanisms can go to work stitching up the break or patching it with a homologous template, in the process creating a knockout or knock-in mutation.
Today, two other, more facile genome-editing techniques have largely overtaken ZFNs. Transcription activator-like effector nucleases (TALENs)—with distinct DNA-recognition domains fused to FokI—appeared in 2011. And in 2013, the field was introduced to the CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein-9), which uses a short guide RNA (gRNA) to target the Cas9 nuclease to its complementary genomic DNA sequence, allowing it to effect a double-stranded break similar to ZFNs and TALENs, but without the protein-design work.
With each new technical advance, genome editing becomes ever more accessible. Yet it’s still not a trivial matter to create a homogenous cell line with a targeted disruption, and even less so with a gain-of-function mutation. Some time- and expertise-limited researchers have thus turned to commercial companies and university core facilities, practiced in the art and armed with the most up-to-date knowledge, to help. Should you opt for one of those services, here’s what you can expect.
What’s involved?
Although the newest kid on the block, CRISPR services are becoming a standard offering, supplementing or even replacing other editing modalities. Yet whichever technology is involved, the basic steps and principles are essentially the same: target selection, reagent design and finally, the editing process itself.
GenScript, for example, starts with a very careful gRNA design to “make sure that we only pick gRNAs that don’t target other regions of the genome,” explains Maxine Chen, marketing liaison for GenScript’s Discovery Biology Services. A ready-to-transfect gRNA vector—Cas9 can be encoded on the same or a separate vector—is prepared for the lab to use, or it can contract for GenScript to do more.
“Making the actual genome-edited cell line is still challenging for labs, and it’s also very time- and labor-intensive,” Chen says. Researchers need to transfect in Cas9 and the targeting gRNA, then make a stable cell line that expresses them. “That includes selection, validating via Sanger sequencing to make sure you have the right sequence and making sure you have a homogenous clone,” she says.
Giving the customer the choice of services to contract for is typical. The Harvard Stem Cell Institute (HSCI) iPS core facility, for example, breaks its TALEN and CRISPR services into three modules—Module 1 is construction and testing of the vectors; Module 2 is gene knockout; and Module 3 is gene-mutation introduction or gene repair. Xin Jiang, the facility’s genome-editing manager, explains that Module 1 is being further divided into submodules for those who provide their own vector but still want it tested. “Or they can bypass the module entirely.”
Sigma-Aldrich’s Cell Design Studio—with custom ZFN and CRISPR services—can offer “any portion of the workflow,” says product manager Erika Holroyd.
Which system is best?
The consensus is that it’s easier and faster to design and manipulate genome-editing reagents for CRISPR system than for ZFN or TALEN. But, especially when outsourcing the work to someone else, that doesn't necessarily make it the better choice. Service providers typically help customers weigh the various factors that may be of import and then “let the project decide which is best,” says Holroyd.
For example, ZFNs and TALENS have been around longer and are better established, and the intellectual property on ZFNs is clear, she notes. “Pharma companies like ZFNs for those reasons.”
For a variety of reasons—often because of some quirk of the local sequence—one particular genomic region may be easier or more difficult to target with one editing reagent than another. And some cell lines may be more recalcitrant to transfection with some reagents than others.
For Sigma-Aldrich, it’s tough to predict a priori which reagent would be the most cost-effective, and Holroyd is reluctant to discuss specifics. “Every project we take in, we go through the details and then price accordingly,” she says. But the HSCI iPS core discloses its prices on its website, charging Harvard and HSCI faculty $1,970 for Module 1, $13,500 for Module 2 and $15,200 for Module 3 using CRISPR, and significantly more for TALEN. Chen says GenScript charges $8,000 per knockout and $9,000 per knock-in. “This would include everything from start to finish.”
With its 20-bp recognition site, a CRISPR reagent is reputed to be less specific—more prone to generating off-target effects—than TALENs and ZFNs, whose recognition sequences are nearly twice as long. But Shondra Pruett-Miller, director of the Genome Engineering and iPSC Center (GEiC) at Washington University in St. Louis, points out that a lot of data have come out recently showing that in a clonal population “the chances of having a targeted event and an off-target event in the same clone [don’t] seem to be as much of a concern as originally thought. A lot of earlier data was looking at pools of cells,” meaning that while a given population may have exhibited cuts at multiple target sites, this was likely not the case for any given cell.
Still, if this is a concern, providers can often adapt newly published approaches or protocols to their services. For example, a catalytically inactive Cas9 can be fused to FokI, creating an obligate dimer with (presumably) heightened specificity. A host of other innovations have emerged in this rapidly evolving field, as well—innovations that could prove challenging to incorporate into your own research without a lot of costly and time-consuming trial and error.
“We are up to date on the latest and greatest advances in the technologies. We’re set up to do this, and that’s what we do day in and day out,” says Pruett-Miller. “The whole point is so that people can keep asking the questions that they’re most interested in, and they don’t have to become the genome-editing expert.”
Reference
[1] Carroll, D, “Genome engineering with zinc-finger nucleases,” Genetics, 188:773-82, 2011. [PubMed ID: 21828278]
Image: iStockPhoto