In just six years, the word CRISPR has gone from meaning a drawer in your refrigerator to the hottest field in biological science. Even people who’ve never so much as glanced at a copy of Scientific American have probably heard at least something about CRISPR, the naturally occurring set of “DNA scissors” that evolved as part of bacteria’s primordial immune system to help it remember and eradicate invading viruses by keeping a copy of segments from an organism’s DNA.
CRISPR and its seemingly limitless potential for gene editing first burst on the world in 2012, when University of California-Berkeley biochemist Jennifer Doudna and French microbiologist Emmanuelle Charpentier, now a director of the Max Planck Institute for Infection Biology in Berlin, presented the first demonstration of its potential to be programmed to precisely cut any piece of DNA, potentially allowing scientists to add or delete genes in precise locations in any type of cell.
CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, was touted as “democratizing” gene editing, replacing costly, complicated, and messy previous approaches with a method that’s been likened to Microsoft Word for the lab. But CRISPR has its own limitations, including off-target effects, and new gene-editing tools are being developed that improve on the original technology as well as deploying it in ways that go far beyond gene editing.
When most people speak of CRISPR, they’re referring to CRISPR-Cas9. CRISPR is the part of the system that recognizes DNA sequences, and Cas (CRISPR-associated proteins) are the set of enzymes that do the actual cutting. But Cas9 is just one of these enzymes—and more are being developed all the time.
The limitations of first-gen CRISPR technology were primarily in specificity.
“The limitations of first-gen CRISPR technology were primarily in specificity,” says Jon Moore, CSO and head of translational science for Horizon Discovery, which has used CRISPR to build a library of over 2,000 edited cell lines. “Today, groups around the world have mutated Cas9 either deliberately or via clever screens in bacteria, to come up with high-fidelity variants with much greater specificity. We have also seen variants like CRISPRi (CRISPR interference) and CRISPRa (CRISPR activation) developed, which allow the behavior of cells to be changed via altering gene expression patterns without mutating their DNA.”
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In February 2018, Broad Institute chemical biologist David Liu reported in Nature on xCas9, a modified version of Cas9 he and his team coaxed to cut DNA in a much broader range of sites than the original Cas9 and which they found to also be much less likely to slice and dice the DNA in unintended places.
Dr. Liu also pioneered DNA base editing, a modification of CRISPR that fuses a "dead" Cas9 (dCas9) to enzymes that can change one nucleotide into another, creating a system that opens up DNA at the correct spot without cutting it, but drives the conversion of say a C to a T. This can correct some of single point mutations implicated in many human diseases in a way that reduces the potential damage that could occur when CRISPR-Cas9 is used to cut and splice in new sequences.
“This is perhaps the most exciting thing of all, because of its potential impact in rare disease,” says Dr. Moore. “It allows for fixing mutations without making a double-stranded break, which would give cells the opportunity to repair the edit into something you don’t want.”
Making it Easi
Another technical challenge for CRISPR is the insertion of foreign DNA cassettes at the cut sites. The commonly used double-stranded DNA donors were only about 10% efficient at best in creating knockout and knock-in mouse models. But in a collaborative study published in Genome Biology in May 2017, scientists from the University of Nebraska and Tokai University in Japan reported that their new system, dubbed Easi-(Efficient additions with ssDNA inserts)-CRISPR, could achieve efficiency as high as 100% by using long single-stranded DNAs as donor cassettes.
Taconic Biosciences announced in April 2018 that it had acquired a worldwide, non-exclusive license to generate rodent models using Easi-CRISPR.
“With Easi-CRISPR, you can start to use CRISPR to not only target areas of the genome, but make insertions of sizeable pieces of DNA—in our experience, up to 4,000 base pairs. This is far easier to set up and execute and can be done in as little as six months, as compared to about a year for homologous recombination. It’s brought greater efficiencies, ease, and speed to the generation of a number of model types,” says John Couse, vice president of scientific services at Taconic, which provides gene inactivation, gene mutation or replacement, transgene expression, RNAi, and gene editing via CRISPR-Cas9, pronuclear injection, and homologous recombination technologies.
From therapy to detection
Gene editing is commonly thought of as a therapeutic tool, but some of the field’s biggest names—including Jennifer Doudna—have launched a new company to develop CRISPR-based diagnostic tools to detect diseases from Zika and malaria to tuberculosis and various forms of cancer. Mammoth Biosciences’ prototype tool leverages CRISPR’s programmability via gRNA to get down to a single base pair resolution level to detect not only a particular disease, but a specific strain of that disease. “I’m excited about the idea that CRISPR has its strongest power as a search engine,” says Trevor Martin, Mammoth’s CEO.
Mammoth is focused on two headliner proteins, Cas12a (a DNA detector) and Cas13a (an RNA detector). “The gRNA programs the CRISPR protein to search for the complement to that guide,” explains Dr. Martin. “If it’s found, that activates a unique property of the protein that cuts many orders of magnitude more of these reporter molecules, which release a color when cut—and that gives you a color readout in the solution.”
The “north star” that Mammoth ultimately envisions is home-based diagnostic testing—a rapid test using a credit-card sized disposable strip, which can then be photographed and uploaded to the company’s smartphone app for remote analysis, confidential results, and professional advice. “The technology also has really exciting implications in the emerging world, because of its low-cost nature and simplicity,” Dr. Martin says. “There are so many things you can detect and want to make sure we use it in the most impactful way possible, zooming in on where this way of approaching detection can have the maximum impact.”
Scaling up
Although it has become fairly routine to use CRISPR technology on a smaller scale in most labs, challenges of reproducibility remain, with differences between users, labs, and cell lines. Two former SpaceX engineers, Paul and Michael Dabrowski, founded a startup called Synthego that aims to make CRISPR more trackable, reproducible, and scalable. In May 2017, it announced its new CRISPR guide design tool, which allows researchers to choose a gene of interest from a curated list of over 100,000 genomes representing more than 9,000 species. The tool uses built-in algorithms to generate guide designs and recommendations for the most efficient guides with low off-target effects and highest likelihood to knock out the function of a gene.
“Onesies and twosies are great for a small academic lab that wants to build a single model of disease, but reproducibility and scalability are ultimately essential,” says Kevin Holden, head of synthetic biology. “Our factory generates the guide RNA fragments using a chemical synthesis that takes days instead of weeks, and following that, we can do the CRISPR manipulation on an automated scale. This enables people to do state-of-the-art CRISPR in their lab, using our resources to automate the process and spend less time doing the experiments and more time thinking about what the results mean and planning the next step. How can you design the best location to do the actual edit relative to what you’re trying to make? We have a tool for that. How can you quickly get information out of these edits to understand what we’ve done in the cell? We have a tool for that. We can partner with you to bring standardization and reproducibility to your processes, whether you’re an academic lab using this technology or a company interested in screening gene knockouts to identify drug targets.”
This month, Synthego launched its Engineered Cells Portfolio, which includes Knockout Clones and Pools, and Advanced Cells. While the Knockout Clones product enables one-click ordering of any human cell line with a guaranteed CRISPR knockout, the Pools product will guarantee at least 50% reduction in protein expression of the gene target. Designed to eliminate the hurdle of learning new methods and optimizing protocols, these products allow scientists to focus on quality results. With Advanced Cells, researchers can leverage Synthego’s CRISPR expertise to design and execute complex cell modification projects, including knock-ins. According to the company, the launch of this portfolio will help scientists accelerate research timelines and ultimately, realize the power of CRISPR, by guaranteeing consistent and predictable results – something that is not currently available on the market.