A research team at Rice University led by bioengineer Gang Bao is working to reveal potential threats to the efficacy and safety of therapies based on CRISPR-Cas9 gene editing technique, even as they appear to be working as planned. Bao's group point out in a paper published in Science Advances that while off-target edits to DNA have long been a cause for concern, unseen changes that accompany on-target edits also need to be recognized and quantified.

Bao is a strong proponent of using CRISPR-Cas9 to treat sickle cell disease, and he is close to getting a cure. Now his team fear that large deletions or undetected changes due to gene editing could persist in stem cells as they divide and differentiate, and have long-term implications for health. His lab developed a procedure that uses single-molecule, real-time (SMRT) sequencing with dual unique molecular identifiers (UMI) to find and quantify unintended large deletions (LDs) along with large insertions and local chromosomal rearrangements that accompany small insertions/deletions (INDELs) at a Cas9 on-target cut site.

The team's tool, called LongAmp-seq (for long-amplicon sequencing), accurately quantifies both small INDELs and LDs. Unlike SMRT-seq, which requires the use of a long-read sequencer often only available at a core facility, LongAmp-seq can be performed using a short-read sequencer. To test their strategy, the team used Streptococcus pyogenes Cas9 to edit beta-globin (HBB), gamma-globin (HBG) and B-cell lymphoma/leukemia 11A (BCL11A) enhancers in hematopoietic stem and progenitor cells (HSPC) from patients with sickle cell disease, and the PD-1 gene in primary T-cells. They found large deletions of several thousand bases at high frequency in HSPCs.

For more details, read the article in Rice University News and Media Relations.