Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
Unless you’ve been hiding under a rock the past few years, you’ve heard about CRISPR-Cas9, the genome-editing tool that’s taken the world by storm.
Researchers can use CRISPR-Cas9 to create or repair mutations, insert reporter genes or purification tags, knock out genes and more, both in cells and in vivo. They can study disease etiology, create drug-discovery models, develop better crops and eradicate pests. And that’s just the beginning; in a recent feature on the technology in The New York Times Magazine, an unnamed scientist says: “[CRISPR] has made so many experiments possible—it’s like standing in a candy store and knowing that you can choose just three things. Meanwhile, there are a thousand more experiments that you wish you could try, if only you had the time.”
Key to the power of CRISPR-Cas9 genome editing is its simplicity. Earlier technologies, such as zinc-finger nucleases (ZFNs) and transcription activator-like effect nucleases (TALENs), offered similar feature sets but were relatively complicated and expensive to use. And they could not easily be multiplexed, meaning researchers could make edits one at a time only.
CRISPR, though, supports expansive multiplexing and requires nothing more complicated than a short guide RNA (gRNA) complementary to the targeted DNA. All users need to do is deliver that RNA into cells, plus the Cas9 nuclease that actually edits the DNA, and the cell takes care of the rest. The reagents already are widely available from such companies as MilliporeSigma, Thermo Fisher Scientific and Addgene.
Though the technology is technically simple, it’s not necessarily practical for some researchers. Some labs lack the expertise to clone single cells—a key step in selecting and expanding properly edited cells from a population. Others lack the personnel or the bandwidth. Whatever their rationale, though, these labs are not out of luck. A number of genome-editing service providers now exist to take up the slack.
Services rendered
Genome-editing companies offer multiple services, but generally speaking, they fall into two categories: reagents and edited cells.
The Harvard Stem Cell Institute (HSCI) iPS Core Facility, for instance, which offers both TALEN- and CRISPR/Cas9-based editing, offers three levels of service, says Xin Jiang, the lab’s genome-editing manager. Module 1 is reagent design, in which the lab reviews the customer’s intended target sequence(s) to identify and design appropriate gRNAs; Module 2 uses those reagents to create a gene knockout in cells, and Module 3 uses them to drive such changes as creating or repairing point mutations or inserting a reporter gene.
Turnaround time averages six months, Jiang says, depending on the cell line, and the cost runs from $1,970 (Module 1) to $15,200 (Module 3) for Harvard/HSCI faculty, or $2,140/$19,100 for external investigators.
At the hESC/iPSC Core Facility at Yale University School of Medicine, cell editing costs $6,500 for Yale faculty or $8,000 for external researchers, and it can be completed in as little as two months, according to lab director Caihong Qiu. At Axol Bioscience, a company that creates, differentiates and (in collaboration with Horizon Discovery) edits induced pluripotent stem (iPS) cells, editing services run from $10,000 to $30,000 and require a minimum of 10 weeks, says chief business officer Sanj Kumar. Axol’s complete service—creating an iPS line, editing that line and differentiating it into some downstream cell type, can take much longer as that is due to the biology and QC needed, Kumar says.
Of course, these time lines are simply estimates, as every cell line and project is different. Some cells grow faster than others, for instance, and some contain more than the usual two copies of every chromosome, says Shawn Shafer, functional genomics market segment manager at MilliporeSigma. One colon-carcinoma line, he notes, is partially decaploid, with 10 copies of some genetic material. “If you want to knock out a particular gene, you may need to knock out 10 copies, which may require subsequent rounds of targeting,” he says.
Other services also are available. For instance, Thermo Fisher Scientific offers a gRNA library production and screening service, says Jon Chesnut, the company’s senior director of research and development. The company has created lentiviral gRNA particles targeting every human kinase (the “kinome”), for instance, but custom gene target gRNAs also can be produced and screened, Chesnut says. And MilliporeSigma can create cell lines that stably express Cas9 for screening applications—just add gRNA—as well as pre-made formalin-fixed paraffin-embedded samples of edited cells to serve as diagnostic standards.
Some companies even offer services based on the many Cas9 variants researchers have devised. MilliporeSigma, for instance, can produce cells expressing a fusion of catalytically inactive (“dead”) Cas9 with the p300 histone acetyltransferase—a reagent that can be used to control gene expression at defined genomic loci. According to Shafer, users of that service need only define precisely where they want the dCas9-p300 expression construct itself to go. “We have identified safe-harbor regions of the genome that don’t interfere with other gene expression,” he says, including the AAVS1 locus in human cells. MilliporeSigma also offers genome-editing services based on ZFN technology.
A question of quality
If you’re outsourcing your genome-editing work, be sure to ask precisely what deliverables you will receive and the quality control checks they will be subjected to. Yale’s genome-editing service, for instance, offers only minimal quality control—they sequence the targeted region to ensure the desired change was made, Qiu says. But the lab also supplies customers with three clones instead of one, to minimize problems from any potential off-target effects.
At HSCI, edited cells are both sequenced at the edit site and karyotyped to ensure the cell’s genome remains grossly intact. For a fee, researchers can request such additional services as pluripotency testing via qPCR and immunostaining.
Similarly, Thermo Fisher Scientific can supplement its default QC of on-target locus sequencing with sequencing of related off-target sites, whole-exome sequencing, protein expression analysis and even assays such as cell morphology and ion flux, says Chesnut.
Genome-editing services are used across the board, from academia to industry. Many clients may have the expertise and money to perform these experiments in-house, but they choose not to, says David Piper, Thermo’s director of research and development. “It’s like anything else. Washing your car is easy, but do you do that? It’s a balance of resources, skill sets and time.”
And indeed, service providers will work with customers to figure out what is and is not compatible with the client’s budget. At MilliporeSigma, for instance, where turnaround time runs 12 to 24 weeks, researchers can save money (and time) by accepting a heterogeneous pool of modified cells that they then must clone themselves (starting at $9,000), vs. having the company clone the cells ($32,000+).
That’s a hefty price tag, Shafer acknowledges. But, he adds, when it comes to outsourcing, you get what you pay for. After all, some clients come to the company only after banging their heads against the wall for months in pursuit of a challenging edit. “$30,000 to $40,000 is expensive,” he says. “But compared to a year of wasted time, it begins to sound much more reasonable.”