Scientists and doctors can better evaluate precision genome editing technology using a new method made public today by St. Jude Children’s Research Hospital. Significant amounts of time and resources devoted to improving CRISPR gene editing technology focus on identifying small, off-target sites that pose a safety risk, which is also a technical challenge. St. Jude researchers solved the problem by creating Circularization for high-throughput genome-wide analysis of nuclease effects by sequencing base editors (CHANGE-seq-BE), an unbiased, sensitive, and resource-efficient method for finding these off-target modifications. It has outperformed conventional approaches and has already been used to support clinical work. The technique was published in Natural biotechnology.
While traditional genome editing technology uses CRISPR-Cas9 to cut a small segment 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 base editors, an important class of precise editors in the CRISPR genome. This is a simple, streamlined way to understand overall core editor activity that allows researchers to select highly specific and active combinations of editors and targets for research or therapeutics. »
Shengdar Tsai, PhD, corresponding author, St Jude Department of Hematology
CHANGE-seq-BE is already being adopted to support clinical research. The article published today includes a case study of an emergency application for the Food and Drug Administration (FDA) for a core editor treating CD40L-deficient X-linked Hyper IgM (X-HIGM) 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% target specificity from the base editor used, with no significant off-target activity, providing valuable safety data to help advance patient treatment.
“This was a really exciting application to support an emergency request to the FDA to treat a patient quickly,” Tsai said. “This illustrates how this method enables rapid understanding of what these editors are doing in the genome and helps advance promising active and specific therapies.” »
Combining efficiency with an unbiased approach yields better results
Tsai’s lab created CHANGE-seq-BE because conventional methods for assessing the security of base editors had to choose between comprehensive coverage and efficient use of resources. Some techniques to comprehensively and unbiasedly find off-target base editing activity require full genome sequencing, which can be extremely expensive and time-consuming. Alternatively, some techniques prescreen suspected off-targets to perform less sequencing and save resources, but these biased techniques can never detect unexpected off-target changes. St. Jude scientists 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 sequencing it immediately, scientists divide the genome into tiny circles of DNA. They then take these circles and expose them to the base editor being tested. Then they treat the DNA with a special enzyme that detects whether base modification has occurred, opening those – and only those – circles of DNA with evidence of base modification into linear strands. The linear strands of DNA are then selectively sequenced, requiring far fewer resources than competing techniques. They optimized it for the two main types of base editors (adenine and cytosine base editors). After developing the method, the scientists wanted to know whether it was actually both more comprehensive and more resource-efficient than conventional approaches. So they tested them face to face.
“When we compared it directly to other methods, CHANGE-seq-BE found almost all of the sites designated by those methods, as well as many sites that it was exclusively able to detect,” Tsai said. “We showed that this unbiased approach was more sensitive using only about 5% of sequencing reads. »
Given the sensitivity of the technique, ease of use and efficient use of resources, others have already started to adopt it. Comprehensive 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 reported in the article, clinical trials at St. Jude and beyond have incorporated the technique into their planning, using it as a tool to evaluate safety and effectiveness. CHANGE-seq-BE was also recently used to characterize the first patient-specific assay alive genome editing processing. Basic research labs studying base editing have also begun using it to test for off-targets early in their process, better identifying the most promising approaches to pursue than existing screens. These early adopters demonstrate the technique’s appeal to researchers and clinicians, and its promise to advance the future of core editing.
“We have enabled those developing these therapies to quickly understand and find the core editors with the highest potential for activity and specificity,” Tsai said. “We hope that methods such as CHANGE-seq-BE will open the door to more genome editing therapies developed for and reaching patients who need them.” »