By Helen Floersh
It took five years to sequence the first human genome. Today, it takes less than 24 hours.
Rapid genome sequencing is one of the many advances indebted to CRISPR-Cas9, the powerful gene editing technology that has fundamentally changed research and biomedicine in the 10 years since its advent. In a new review published Jan. 20 in Science by CRISPR pioneer Jennifer Doudna, Ph.D., and Doudna lab doctoral candidate Joy Wang, the researchers lay out the past, present and future of the technology as well as the many challenges it must surmount to reach its full potential.
CRISPRs—an acronym that stands for clustered regularly interspaced short palindromic repeats—are short sequences of DNA that are commonly found in the genes of microbes alongside proteins, dubbed CRISPR-associated proteins, or Cas. Since the late 1980s, researchers have been studying how RNA molecules transcribed from the CRISPR DNA sequences instruct Cas proteins on where to cut strands of DNA, essentially acting as a pair of molecular scissors.
In June 2012, Doudna and her colleagues at Stanford University published a groundbreaking paper that described how to use the CRISPR-Cas9 protein and its accompanying RNA to edit bacterial genes. In the decade since, CRISPR-Cas9 has become the most widely used form of the technology and has “profoundly changed biological research,” Doudna and Wang wrote in their new review.
For instance, CRISPR-Cas9 has replaced the once-tedious process of removing or editing genes in mouse models. Instead of a year to produce genetically modified mice, it now takes as little as four weeks. The technology has dramatically altered the efficiency of preclinical research, as “the production of [knockout] and transgenic mice has now become routine for research applications,” Doudna and Wang wrote in their review.
CRISPR-Cas9 has also given researchers and clinicians the ability to screen genes rapidly and obtain “clinically actionable” information in a matter of days, the scientists added. Furthermore, it can now be used to alter multiple DNA sequences or base pairs at once in a technique called multiplex editing. While multiplexing has predominantly been used for editing plant genes to create new types of crops and increase crop yield and quality, it could have many applications in biomedicine, too.
One example is the creation of pigs without a set of viruses known as PERVs, or porcine endogenous retroviruses. Although pig organs are considered a viable solution to the shortage of organs for transplant in humans, PERVs could lead to rejection. To get around this, researchers used CRISPR-Cas9 to inactivate PERV genes and reduce viral transmission to human cells. In 2017, a company called eGenesis raised a US$34 million series A to build upon the work and added a $100 million series B in 2019.
Multiplexing has more to offer beyond cutting and inserting genes, the researchers wrote, and will require advances such as base editing. If traditional CRISPR-Cas9 technology is molecular scissors, base editing is an X-Acto knife, a precision tool to permanently alter a single base pair without breaking the DNA. This solves a major problem for multiplexing, which makes multiple DNA strand breaks simultaneously and can thus lead to DNA damage-response mechanisms that ultimately result in cell death.
Base editing’s precision makes it ideal for tackling conditions that arise from a single point mutation and lead to loss of function in genes, such as in Hutchinson-Gilford progeria syndrome. In a preclinical study published in early 2021, researchers described how they corrected the mutation that leads to the disease in mouse models, nearly doubling the animals’ lifespans from 215 to 510 days. Verve Therapeutics is already using CRISPR-Cas9 base editing in a clinical trial for familial hypercholesterolemia.
Base editing is being tested in sickle cell disease, too. Beam Therapeutics is studying BEAM-101 in its BEACON trial, which dosed its first patient last November. BEAM-101 works by altering a gene so it activates fetal hemoglobin, thereby compensating for the dysfunctional hemoglobin that leads to sickle cell disease symptoms. Trial data from the first patient cohort are expected next year.
But to truly see progress in using CRISPR systems to treat disease, delivery mechanisms and manufacturing will need to be more efficient and cost effective, Doudna and Wang wrote. Viral vector delivery, one of the most popular methods, is difficult to scale, they noted. Delivering the therapy with autologous hematopoietic stem cells—Beam’s method for BEAM-101—is also pricey and resource-intensive. And that’s before the many costly regulatory hoops agencies like the FDA require to ensure safety.
The researchers worry that when CRISPR therapies do come on the market, patients will suffer from sticker shock. “Even if a treatment passes through all clinical trial phases and gets FDA approval, the potential retail price charged to cover manufacturing costs may be unaffordable to most patients without changes to the current healthcare infrastructure,” Doudna and Wang wrote.
Researchers are working on solutions, they added. One that scientists are particularly excited about is extracellular vesicles and viruslike particles, which work by using the viral envelope and structural proteins that can get into cells but without actual viral genetic material. These combine viral vectors’ best characteristics with synthetic material-based delivery, like lipid nanoparticles, and have favorable safety profiles, because they lack the ability to integrate with the genome or stick around too long in the cell.
While it remains to be seen how delivery, manufacturing and pricing will play out, Doudna and Wang predict that the next 10 years will see CRISPR technology become faster and cheaper to use for applications like genome sequencing. They also anticipate that it will be paired with machine learning and live cell imaging both in the clinic and in research settings. The technology will be used in more applications to treat more diseases and perhaps even to prevent them. If safety and efficacy are established, “genome editing might become a prophylactic against neurodegenerative or cardiovascular disease,” the researchers wrote.
“Such opportunities would require detailed knowledge of the genetic of multigenic disease and the means to deliver to organs including the brain and heart—neither of which are small tasks,” Doudna and Wang wrote. “But the potential benefits may drive innovation in these areas beyond what is possible today.”