Research led by the University of Verona and the University of Harvard shows the importance of taking human genetic diversity into account when designing gene editing therapies for conditions such as sickle cell disease.
A number of different CRISPR genome editing techniques are currently being developed to treat conditions such as β-thalassemia. “Although a variety of in vitro and cell-based experimental methods can be used to empirically nominate off-target sites, these methods either use homology to the reference genome as a criterion to define the search space and/or use a limited set of human donor genomes to evaluate off-target potential,” write Luca Pinello, a researcher at Harvard Medical School, and colleagues in Nature Genetics.
“Therefore, computational methods may be especially useful to predict the impact of off-target sequences not found in reference genomes.”
The researchers have created a computational tool called CRISPRme to assess the impact of single-nucleotide polymorphism (SNP) and indel genetic variants on potential off-target sites for CRISPR-based therapies.
Pinello and team tested the software with a BCL11A enhancer targeting guide RNA that is being tested in clinical trials for sickle cell disease and β-thalassemia at the moment. They found that a gene variant common in African American populations was the most likely off target site for this guide RNA.
The researchers calculated that up to 10% of sickle cell disease patients with African ancestry could carry the risk allele they identified, which could lead to rates of up to 10% cleavage at an off-target site not previously identified as problematic.
“Gene editing efforts that focus on a specific patient population should consider genetic variants enriched in that population during off-target evaluation,” write the authors. “However, our analysis also shows that variant off-targets may be private to a given individual, so all humans could potentially be susceptible to such an effect.”
The lack of diversity in early genetic studies and reference genomes has proved problematic, as it means many clinical genetic tests are now most relevant in White, European populations. This research highlights that investigators also need to be aware of this potential issue in newer therapies under development such as those using CRISPR-Cas9 or similar methods.
“We expect that variant-aware of-target assessment will become integral to therapeutic genome editing evaluation and provide a powerful approach for comprehensive off-target nomination.”