Since its introduction in 2013, the use of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) 9 system for genome editing has exploded. Products for CRISPR/Cas9 range from plasmids and oligonucleotides to transgenic animals and screening services. According to Bioinformatics LLC’s report, “The Market for CRISPR/Cas9 Genome Editing Products,” published in May and based on a survey of more than 550 scientists, labs are planning to increase their CRISPR/Cas9 budgets 37% over the next 12 months. The estimated $200 million market has grown so quickly due to the technology’s accessibility and rapid development, and new applications. As a result, CRISPR/Cas9 is displacing other genome editing techniques and expanding genome editing to new users, promising continued robust growth.

Compared to other genomic editing techniques, CRISPR/Cas 9 is simpler, more flexible and lower cost (see IBO 6/15/15). Originally found in bacteria, the CRISPR pathway and Cas9 protein form a double-strand break at a DNA sequence site, which must be next to a PAM (protospacer adjacent motif) sequence. All that is required to perform a CRISPR/Cas9 experiment is a guide RNA (gRNA) (a fused CRISPR RNA and tracer RNA) to locate the target sequence and the Cas9 nuclease to make the cut. The cut can be repaired by nonhomologous end joining for insertions or deletions, a so-called knock out; or by homologous recombination (HR), which introduces a specific mutation into sequence, a so-called knock in; and other insertions such as a fluorophore for labeling. Unique advantages of the CRISPR/Cas9 system include the ability to use multiple gRNA to alter several genes simultaneously and that it works in most organisms.

Key to the market’s development has been the technology’s accessibility, specifically plasmids and oligonucleotides. Not only are the building blocks of a CRISPR/Cas9 experiment familiar to most biologists, but they are also readily available and affordable. Addgene, a nonprofit plasmid repository, is a key supplier of plasmids for academic research, including CRISPR/Cas9 plasmids. Researchers deposit their plasmids with Addgene, which performs quality control, stores them and distributes them to scientists. The cost of a single plasmid is $65, but this price decreases with volume. The organization has shipped over 540,000 of all plasmid types since 2004.

Addgene is a major supplier to academic researchers for CRISPR/Cas9. “[Early scientists in the field] deposited from those first few papers, almost before publication, so that when the papers came out, the materials were ready,” explained Joanne Kamens, Ph.D., executive director of Addgene.

Addgene offers more than 100 CRISPR plasmids from 50 depositors. “Since January of 2013, when the first paper came out, we’ve probably shipped over 45,000 plasmids,” said Dr. Kamens regarding CRISPR. “Some of our most popular plasmids are those basic tools needed for a CRISPR experiment. The first one being the empty gRNA expression vectors, which allow scientists to clone the gRNA targeting their gene of interest,” said Caroline LaManna, PhD, head of Outreach for AddGene. “The second one is the Cas9 expression vectors.”

Addgene has been the first provider to make available the latest advancements in CRISPR/Cas9 tools, including those addressing off-target effects. Among these are Cas9 nickases, which “nick” a single DNA strand so that two targeting CRISPRs with two separate gRNAs can be used for greater accuracy. “Cas9 variants allow scientists to choose the function that best fits their experiment,” noted Dr. LaManna. “We have plasmids for Cas9 to generate double-strand breaks in many organisms, or variants to express the nickase Cas9 for generating single-strand breaks, or for tagging a gene and a variety of other variants.”

Addgene has also contributed to the technology’s development by providing technical resources and support, according to Dr. LaManna. “We’ve created a community of people working together to share. Our mission is to accelerate research by facilitating the rapid dissemination of materials and data.” As Dr. Kamens noted, “We’re crowdsourcing, essentially. All these different depositors, they want their materials to get out into the community.”

Addgene also supplies pooled libraries of plasmids. “Pooled libraries are used essentially for ‘fishing’ experiments. Scientists screen for a certain outcome using a library and, in this way, identify a small number of genes that have a role in that outcome,” explained Dr. Kamens.” Addgene currently offers 15 pooled libraries with more in process. As Dr. Kamens told IBO, Addgene is working to optimize use of these libraries. “That is a resource that often goes into the freezer and then is wasted and never used again. We’re trying to work on technologies and a service so we’ll be able to reamplify and distribute these materials to the community.”

Compared to Addgene, Sigma-Aldrich provides a wider range of CRISPR/Cas9 products and services. Although many are the same basic components that Addgene and other nonprofit resources supply, such as bioinformatics tools for designing gRNA, the company does not view the technology’s simplicity or accessibility from other providers as a drawback. As Shawn Shafer, PhD, Functional Genomics manager at Sigma, told IBO, “The more people that are adapting the technology, kind of getting their feet wet, the more potential future customers we have.”

As a commercial provider with a history in genetic editing, serving both academic and industry researchers, Sigma has contributed CRISPR/Cas9 market growth and technology advancement. Sigma’s products include optimized plasmids and delivery systems, designed to save time and labor and enable broader adoption of CRISPR/Cas9. “We offer the transfection reagents in all those different combinations, with Cas9 and gRNA together, separately, with and without fluorophore selection markers,” explained Dr. Shafer. Sigma has steadily expanded its offerings, adding mRNA for gRNA and Cas9 delivery. “RNA is ideal for microinjection for creating transgenic animals. Plasmid DNA has the potential to integrate in the genome, but RNA does not,” he said. “So that has kind of changed the way the transgenic animal generation is done.”

Experience gained from the company’s development and commercialization of other genome editing techniques, such as zinc finger nucleases (ZFNs), has accelerated its development of CRISPR/Cas9 products. “[W]e have this legacy knowledge, and all of it has been applicable. Everything we learned with ZFNs about off targeting, about design, it’s all applied to CRISPR,” said Dr. Shafer. The same is true for Sigma’s work with lentiviruses, according to Dr. Shafer. Sigma’s lentivirus can be used for difficult-to-transfect cells and screening applications. “Lentivirus integrates into the genome permanently, and so it will continuously express the nuclease and the gRNA, which makes it easier to enrich for cutting, whereas with transfection, those reagents get degraded and go away,” he explained.

Among Sigma’s most popular offerings are custom products for CRISPR/Cas9. “A couple of those things that we get a lot of requests for are donor design and then lentiviral manufacturing,” said Dr. Shafer. “Sites like Addgene offer all these constructs as DNA, but if you want to turn them into a virus, you’re going to have to make it yourself or find a provider,” he added.

As for continued technology and market progress, Dr. Shafer highlighted advances in DNA synthesis. “One of the things that is a big area is introducing new sequences, putting in new DNA to see what happens,” he explained. “So now a lot of people are just getting these plasmids synthesized de novo, synthesized from scratch, instead of doing classic cloning, the piece-the-puzzle-together approach, which is time consuming and can be frustrating.” He also expects further developments to address HR repair efficiency. “A lot of people are focusing on ways to increase the rate of homologous-directed recombination because it happens at very low frequency.”

Founded in 2005, publicly held Horizon Discovery is primary focused on CRISPR/Cas9 services, such as gRNA and donor design, and reagent validation, but also offers a wide range of tools, such as vectors and knockout cell lines. In contrast to many other CRISPR/Cas9 tool providers, Horizon specializes in service offerings, another facet of the CRISPR/Cas9 market’s growth, and one that holds particular promise for drug development.

Describing Horizon’s service offerings, Eric Rhodes, PhD, CTO and vice president of R&D, told IBO, “[I]f you are not confident to do it yourself, we will do the genome engineering ourselves in a cell line of choice. We can also offer to do that in animals—in mice, rats and rabbits,” he explained. “We go beyond that in terms of we can create, for example, reporter lines that people use in screening. . . . We can use that line in screening, [and] we can give you screening results.” The company’s most popular CRISPR offerings are cell-line creation and animal transfection. “A year ago, more than 50% done [of Horizon’s work] was with things other than CRISPR. Now, more than 50% is done with CRISPR.”

Dr. Rhodes expects services to become more important as CRISP/Cas9 technology advances. “If you are looking at doing multiple genes or lots of cells or libraries, then I think service providers are going to become more and more important.” Among the services Horizon offers for drug development using CRISPR/Cas9 are target identification and validation. “We make use of the growing body of patient information relating to genetics and disease to build optimized cellular and animal models for drug screening. We have used gRNA screening to identify new targets in oncology, which hadn’t been seen using other screening methods (like siRNA, for example),” he explained. “We can then further apply CRISPR to explore entire biological pathways to determine what the best targets in those pathways might be for further drug development.”

Horizon has also combined CRISPR/Cas9 with recombinant adeno-associated virus (rAAV), to which it holds exclusive license, to address limitations in reagent delivery, such as larger DNA segments for HR. “RAAV is particularly adept at delivering DNA into the nucleus of many types of cells, which can greatly improve targeting efficiencies,” said Dr. Rhodes. For screening, Horizon will create specialized libraries to study a particularly pathway. “[T]here might be 50 genes in it, and we can make a library that’s specific just to those 50 genes and not have to do the whole-genome screen,” explained Dr. Rhodes.