Scientists and doctors can better evaluate precision genome editing technology using a new method released today by St. John’s Children’s Research Hospital. Jude. Significant time and resources spent on improving CRISPR gene editing technology are focused on identifying small off-target sites that pose a safety risk, which is also technically challenging. The researchers of St. Jude tackled the problem by creating Traffic for high-throughput analysis of genome-wide nuclease effects by sequence-based processors (CHANGE-seq-BE), an unbiased, sensitive and resource-efficient method for finding these off-target changes. It outperformed conventional approaches and has already been used to support clinical work. The technique was published in Nature Biotechnology.
While traditional genome editing technology uses CRISPR-Cas9 to cut a small section of DNA from the genome, scientists have continued to develop more precise versions, including base editors, which can find and replace individual DNA base pairs.
We developed CHANGE-seq-BE to enable scientists to better understand core editors, an important class of precise CRISPR genome editors. It is a simple and improved way of understanding the global activity of key editors that enables researchers to select highly specific and active editor and target combinations for research or treatment.”
Shengdar Tsai, PhD, corresponding author, Department of Hematology St. Jude
CHANGE-seq-BE has already been adopted to support clinical research. The paper published today includes a case study of an emergency application for the Food and Drug Administration (FDA) for a core editing program that treats CD40L X-linked Hyper IgM (X-HIGM) deficiency syndrome. X-HIGM is a genetic immune disease that base editing may be able to correct. CHANGE-seq-BE was able to confirm 95.4% on-target specificity from the base editor used, with no significant off-target activity, providing valuable safety data to help advance patient therapy.
“It was a really exciting application to support an urgent application to the FDA to rapidly treat a patient,” Tsai said. “It exemplifies how this method enables a rapid understanding of what these editors are doing in the genome and helps advance promising active and specific therapeutics.”
Combining efficiency with an unbiased approach provides better results
Tsai’s lab created CHANGE-seq-BE because conventional methods for assessing the safety of core editors had to choose between comprehensive coverage and efficient use of resources. Some techniques to fully find off-target activity of base editing in an unbiased manner require whole-genome sequencing, which can be prohibitively expensive and time-consuming. Alternatively, some techniques preselect off-target suspects to perform less sequencing and save resources, but these biased techniques can never detect unexpected off-target modifications. The scientists of St. Jude designed CHANGE-seq-BE to capture the best of both approaches: a comprehensive solution that would also be resource efficient.
To do this, CHANGE-seq-BE starts with an entire genome, but instead of immediately sequencing it, scientists divide the genome into tiny DNA circles. They then take these cycles and expose them to the core processor under test. They then treat the DNA with a special enzyme that detects whether base editing has occurred, opening those – and only those – DNA circles with base editing elements into linear strands. The linear DNA strands are then selectively analyzed, requiring far fewer resources than competing techniques. They optimized it for both major types of base processors (adenine and cytosine base processor). After developing the method, the scientists wanted to know if it was really more comprehensive and resource-efficient than conventional approaches, so they tested them head-on.
“When we compared it directly with other methods, CHANGE-seq-BE found almost all the sites defined by those methods, as well as many that it was exclusively able to detect,” Tsai said. “We showed that this unbiased approach was more sensitive while using only about 5% of sequence reads.”
Given the technique’s sensitivity, ease of use, and resource efficiency, others have already begun to adopt it. Full experimental protocols and software to enable CHANGE-seq-BE are described in the study, enabling this adoption. For example, in addition to the clinical application mentioned in the paper, the clinical trials at St. Jude and beyond have incorporated the technique into their programming, using it as a safety and efficacy assessment tool. CHANGE-seq-BE was also recently used to characterize the first patient-specific in vivo genome editing therapy. Basic research labs investigating core processing have also begun using it to test off-targets early in their process, better identifying the most promising approaches to pursue from existing screens. These early adopters demonstrate the technique’s appeal to both researchers and clinicians and its promise to advance the future of core processing.
“We’ve enabled those developing these therapies to quickly understand and find the key authors with the highest possible activity and expertise,” Tsai said. “We hope that methods like CHANGE-seq-BE will open the door to more genome editing therapies being developed and reaching the patients who need them.”
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