With the first CRISPR gene editing therapies now in clinical trials there is an increased urgency to be able to quickly identify any unintended and potentially harmful changes that might be made to nontarget stretches of DNA. Scientists at the Helen F. Graham Cancer Center & Research Institute, and the University of Delaware, have developed a new way of identifying even infrequent alterations to regions of DNA near to the CRISPR target much more quickly than existing approaches. Described in Communications Biology, the new tool can detect the different outcomes of CRISPR gene editing in just 48 hours, compared with existing processes that can take up to two months of costly, complicated DNA analysis.
“We’ve developed a new process for rapidly screening all of the edits made by CRISPR, and it shows there may be many more unintended changes to DNA around the site of a CRISPR repair than previously thought,” said Eric Kmiec, PhD, director of Christiana Care’s Gene Editing Institute. “CRISPR will probably never be perfect 100% of the time. But CRISPR tools are constantly improving. And if we can achieve a 70% or 80% rate of precision—and reveal and understand the importance of any changes that occur alongside that repair—that brings us much closer to safely using CRISPR to treat patients. We hope our new tool can help accelerate efforts to achieve that goal.”
Kmiec is principal author of the researchers’ published paper, which is titled, “Understanding the diversity of genetic outcomes from CRISPR-Cas generated homology-directed repair.”
CRISPR—clustered regularly interspaced short palindromic repeats—is a bacterial defense mechanism that can recognize and slice up the DNA of invading viruses. Scientists have learned how to harness CRISPR as a tool for targeting and disabling malfunctioning genes, or for correcting single-base mutations or small deletions that may be the cause of devastating diseases, the authors explained.
“The simplicity of design coupled with the efficiency of activity enables a broad spectrum of clinical development and introduction into patient populations has already begun,” they wrote. Just last month CRISPR Therapeutics and Vertex Pharmaceuticals announced positive safety and efficacy data from the first two patients enrolled in two clinical trials evaluating the CRISPR-Cas9 gene editing therapy CTX001, for sickle cell disease and beta thalassemia.
Kmiec noted that most tools for analyzing CRISPR gene edits are suited to verifying that the repair was successful, and not for revealing any unwanted alterations that might occur to nearby strands of DNA. As the authors pointed out, when researchers report success in using CRISPR to repair malfunctioning genes, they “may inadvertently underreport the collateral activity of this remarkable technology … Thus, as CRISPR-Cas systems advance toward clinical application, it becomes increasingly important to ensure that researchers and physicians can obtain all outcomes of a specific gene editing reaction.” With this information scientists will be better equipped to make “a more educated choice surrounding the types and amounts of genetic engineering tools to employ for the treatment of a genetic disease.”
Screening for unintended CRISPR edits currently requires the extraction and analysis of enormous amounts of cellular DNA. It’s a kind of needle-in-a-haystack process that can take up to two months, and may still not capture all the DNA alterations. Scientists at the Gene Editing Institute found a way around this problem by working with a system they have developed that performs gene edits on circular plasmid DNA extracted from the cell. The researchers found that working in a plasmid or “cell-free” system eliminated a lot of the complex biological activity within a cell that makes it hard to isolate the full array of DNA changes introduced by CRISPR. “We have taken a decidedly reductionist approach to this problem by studying the mechanism of CRISPR-directed gene-editing in a system that employs a mammalian cell-free extract to drive the gene-editing reaction,” they explained. “In this system, we can carefully control the level of reaction components and as a result, we can catalog the distribution of insertions, deletions, and duplications as a function of each set of genetic tools.”
In the reported study, the team said that the system allowed them to “visualize the wide array of genetic modifications created through the process of CRISPR-directed gene editing in a straight-forward and simple fashion.” And because the tool can screen outcomes of an edit quickly and cost-effectively, it frees scientists to execute and screen multiple trial edits, and many more than is practical with a cell-based system. This will make it possible to identify unintended mutations that may occur at relatively rare frequencies and which might otherwise be missed.
The plasmid-based approach also allowed the investigators to test variations of CRISPR editing tools that employ Cas9 or Cas12a, and DNA templates. They found that in their tests, the rate of “precise repair”—repairs that are accomplished without introducing unintended mutations—varied considerably, from 5% to 64%, depending on the enzyme and template employed.
“One important aspect of this in vitro system is that it affords us the opportunity to visualize the wide array of genetic modifications created through the process of CRISPR-directed gene editing in a straightforward and simple fashion,” the scientists stated. “This information is important because it provides clarity surrounding the generation of unanticipated and unintended repair products created by gene-editing tools. Such information forms the basis for determining risk-benefit decisions surrounding the effectiveness of genetic engineering tools to treat human disease.”
Kmiec cautioned that the unintended changes revealed by their work involve “subtle mutations” to DNA around the immediate site of the genome targeted for repair. That’s very different, he said, from the debated concerns about the risk of CRISPR causing off-target mutations distant from the target site, or making random cuts across the genome. “It’s important to note that in all instances we were still seeing CRISPR achieve a fantastic level of successful repairs that would have been unimaginable even five years ago,” stated lead author Brett Sansbury, PhD. “But we saw a lot of other changes to DNA near the site of the repair that need to be better understood so that when we correct one problem, we’re not creating another.”
Sansbury said those changes included deletions, duplications, and rearrangements of DNA code. And while the researchers believe the vast majority of these unintended edits may have no consequence for patients, it’s important to identify them and determine which ones might pose a risk, for example by generating a gene edit that results in a harmful protein being produced. The scientists concluded, “As most investigators, we believe that the mechanistic questions surrounding human gene-editing should be further addressed as they form the foundation for human therapy. Here, we contribute to this effort by providing a more accurate view of the multiple outcomes of CRISPR-directed gene editing in an unbiased and highly validated fashion.”
The work to develop a better way to screen for CRISPR-induced mutations is part of a broader effort at the Gene Editing Institute, which is pioneering advances in editing DNA plasmids extracted from human cells. The team has already has used the cell-free approach to engineer multiple edits simultaneously, and is partnering with industry to develop new approaches to personalized cancer care. Gene Editing Institute researchers have developed a tool that can rapidly reproduce, in a human DNA sample, the complex genetic features of an individual patient’s tumor. Those samples can then be used to screen multiple chemotherapies and other cancer drugs to design a treatment best suited for the individual patient.