Biologists have long been able to edit genomes with molecular tools. About ten years ago, they became excited by enzymes called zinc finger nucleases that promised to do this accurately and efficiently. But zinc fingers, which cost US$5,000 or more to order, were not widely adopted because they are difficult to engineer and expensive, says James Haber, a molecular biologist at Brandeis University in Waltham, Massachusetts. CRISPR works differently: it relies on an enzyme called Cas9 that uses a guide RNA molecule to home in on its target DNA, then edits the DNA to disrupt genes or insert desired sequences. Researchers often need to order only the RNA fragment; the other components can be bought off the shelf. Total cost: as little as $30. “That effectively democratized the technology so that everyone is using it,” says Haber. “It’s a huge revolution.”
Researchers have traditionally relied heavily on model organisms such as mice and fruit flies, partly because they were the only species that came with a good tool kit for genetic manipulation. Now CRISPR is making it possible to edit genes in many more organisms. In April, for example, researchers at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, reported using CRISPR to study Candida albicans, a fungus that is particularly deadly in people with weakened immune systems, but had been difficult to genetically manipulate in the lab3. Jennifer Doudna, a CRISPR pioneer at the University of California, Berkeley, is keeping a list of CRISPR-altered creatures. So far, she has three dozen entries, including disease-causing parasites called trypanosomes and yeasts used to make biofuels
Zinc Fingers (ZFN)
Zinc fingers are transcription factors; each finger module recognizes three to four bases of sequence, and by mixing and matching those modules researchers can more or less target any sequence they wish (with some limitation: Sigma Aldrich, which commercializes ZFN technology, can produce a working ZFN for about every 50 bp on average, says Greg Davis, a Principal R&D Scientist who spearheads genome-editing technology at the company).
A ZFN is a heterodimer in which each subunit contains a zinc finger domain and a FokI endonuclease domain. The FokI domains must dimerize for activity, thus increasing target specificity by ensuring that two proximal DNA-binding events must occur to achieve a double-strand break.
In 2013, according to functional genomics market segment manager, Shawn Shafer, the company offers both custom and off-the-shelf CompoZr® ZFNs for $4,000 to $7,000 apiece. These enzymes are all validated prior to sale, but recently, the company rolled out a “fast ZFN” option for those willing to skip the validation, providing four custom ZFNs for $3,000.
“We know that if we send four pairs of ZFNs for a knockout, 90% of the time at least one will be successful,” Shafer says.
Transcription activator-like effector nucleases, or TALENs, are dimeric transcription factor/nucleases built from arrays of 33 to 35 amino acid modules, each of which targets a single nucleotide. By assembling those arrays just so, researchers can target nearly any sequence they like.
Labs can build custom TALENs for a fraction of what ZFNs cost. Addgene sells individual TALEN plasmids for $65 apiece, and complete kits for a few hundred dollars. Dan Voytas’ popular Golden Gate TALEN 2.0 kit costs $425.
In the CRISPR/Cas9 system (the unwieldy acronym stands for: “Clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated-9”), the Cas9 nuclease makes a double-stranded break in DNA at a site determined by a short (~20 nucleotide) guide RNA. As with other systems, that break can be repaired by NHEJ or homology-directed recombination, depending on how it’s used.
Unlike TALENs or ZFNs, CRISPR/Cas can be multiplexed by adding multiple guide RNAs. Recently, for instance, Rudolf Jaenisch of Massachusetts Institute of Technology and the Whitehead Institute for Biomedical Research demonstrated that he could make five simultaneous mutations in mouse embryonic stem cells using five guide molecules
Recombinant adeno-associated viruses (rAAV)
Recombinant adeno-associated viruses (rAAV) is another method.
Each rAAV line costs $990 for academic users. Alternatively, the company can generate custom genomic modifications for $35,000 and up. Academics can build custom lines themselves if they belong to Horizon’s Centers of Excellence program, through which the company has licensed rAAV technology for noncommercial applications. The company has established 30 Centers, including at the National Cancer Institute, Cambridge University, Johns Hopkins University and the Fox Chase Cancer Center.