Integrated DNA Technologies

Ashley Jacobi, director, applications and market development at Integrated DNA Technologies (IDT) and Gavin Kurgan, bioinformatics applications development manager at IDT, highlight how pioneers in CRISPR innovation can advance the delivery of safer and more efficient genome editing platforms with off-target analysis capabilities.

CRISPR-Cas Gene Editing: The Need for Safety Assessments

CRISPR-Cas systems have revolutionized genetic engineering by enabling precise genome modifications. Derived from a bacterial immune mechanism, CRISPR-Cas9 allows targeted DNA edits in various organisms, offering vast potential in medicine, agriculture and biotechnology. However, these powerful tools come with significant risks. A primary concern is off-target effects, where unintended genomic regions, like the targeting sequence (the guide RNA), are edited. These off-target edits can lead to unwanted genetic changes that disrupt essential genes, potentially causing adverse effects on cellular function, viability and even leading to secondary diseases. Robust safety assessments are crucial to ensuring CRISPR-Cas therapies do not inadvertently cause harm.

Challenges in Assessing the Safety of CRISPRCas Therapies

Assessing the safety of CRISPR-Cas therapies is complex due to several factors. First, the diversity of technologies used for off-target nomination complicates the establishment of clear analytical guidelines. Current methods fall into three categories: in cellulo, in silico and biochemical in vitro assays, each with distinct advantages and disadvantages. For example, cell-based assays are known for high true-positive rates but face challenges in representing diverse genome populations. Biochemical in vitro assays are known to be sensitive but struggle to replicate the cellular environment, impacting enzymatic activity and false positive rates. Computational in silico approaches can quickly predict sites with similarity and from diverse populations, but suffer from high false-positive rates due to insufficient data on enzymatic and biological activity. Additionally, all these methods leverage next-generation sequencing (NGS) for off-target detection which can have limitations in variables ranging from library preparation efficiency to the amount of sequencing reads / number of genomes interrogated from sample-to-sample. These challenges highlight the need for comprehensive information and standardized methods for off-target safety assessment.

“Advancements in off-target analysis capabilities are paving the way for safer and more efficient genome editing platforms, unlocking the true promise of CRISPR and revolutionizing genetic medicine”

Pioneers are Meeting the Moment

Pioneers in the field are actively working to address these challenges, both operationally and technically. Operationally, some of the most successfully de-risked genome editing work has focused on the use of orthogonality in the nomination phase to highlight the strengths of each method while covering weaknesses. An exceptional example of this is in recent work led by the Children’s Hospital of Pennsylvania and Penn Medicine to develop an “N of 1” in vivo base editor treatment for a child with a urea cycle disorder. In this work, in cellulo, in vitro, and in silico methods were all used in concert, considering the patient’s genome in the computational and in vitro nomination of off-targets while using knowledge of true-positive rates derived from in cellulo assays across multiple variables (gRNAs and cell types) to add novel putative off-targets and guide off-target prioritization. To better standardize this sort of process, the FDA has recently issued draft guidelines echoing the recommendation of multiple orthogonal methods for offtarget nomination to mitigate known or unknown weaknesses. Technically, a number of scientific research groups are driving further improvements and standardization to the nomination process of existing or novel technologies. Integrated DNA Technologies recently reported an improved in cellulo workflow called UNCOVERseq, which sensitively nominates off-target sites with defined input requirements and analytical process controls, and demonstrated superior performance compared to existing methodologies. Additionally, new technologies created to perform unbiased nomination of newer base editor modalities, such as CHANGE-seq BE, are being created and explored to help inform recommendations for next-generation editing modalities.

The Future of the Field: Addressing the Unmet Needs

The future of CRISPR-Cas safety assessments looks promising with ongoing innovations and regulatory efforts. Researchers are continuously improving upstream biological and library preparation inefficiencies of different off-target nomination and confirmation methods to drive to lower limits of detection with higher confidence. This effort, in combination with standardization efforts led by the Genome Editing Consortium at the National Institute of Standards and Technology, will help to continuously determine true positives and negatives in variable experimental contexts to better comprehensively benchmark different technologies. As the field progresses, comprehensive guidelines and standardized methods will be essential to further ensure the reproducibly safe and effective use of CRISPR-Cas technologies in clinical applications. It is our belief that part of this standardization also will need to include new standards on how to more uniformly consider the risk of off-targets in the genome editing space, akin to what has been done with guidelines produced for heritable disease and oncology.

The advancements in off-target analysis capabilities are paving the way for safer and more efficient genome editing platforms. By addressing the challenges and unmet needs in safety assessments, pioneers can unlock the true promise of CRISPR and revolutionize genetic medicine.