Study reveals complex on-target genomic rearrangements induced by CRISPR-Cas9

An international team of researchers, led by SciLifeLab Fellow alumna Claudia Kutter (Karolinska Institutet), has discovered unexpected CRISPR/Cas9 on-target effects. Their findings have broadened the consequential spectrum of the system and thus, highlighted the importance of meticulous genomic validation whenever using CRISPR/Cas9. The team also presented a data-driven workflow enabling detailed dissection of the on-target sequence content with superior resolution.

During recent years, the CRISPR/Cas9-system, which also received a Nobel Prize in 2020, has been widely used by researchers across the world. The system allows researchers to modify the genetic content in a cell to study the molecular roles of genes or regulatory elements and to correct disease-causing genetic variants. As more and more researchers utilize the system, more and more consequential side effects have been reported, however.

In a recent study, led by SciLifeLab Fellow Alumna Claudia Kutter (KI), an international team of researchers made puzzling molecular observations after modifying a genetic region containing tRNA genes or other regulatory sequences in the genome of human cancer cells. Instead of reducing cancer cell growth, the cancer cells grew faster. In an attempt to explain their discoveries, the researchers combined some of the latest technological approaches, including Xdrop target enrichment and Oxford Nanopore Technology long read sequencing, with their own, newly developed computational analysis pipeline, Xdrop-LRS. The results uncovered new and unexpected CRISPR/Cas9 on-target effects.

The researchers could observe, in high frequency, varying on-target effects, including target gene duplication, reintegration in different orientations, large deletions, insertion of exogenous sequences, and insertion of clustered interchromosomal fragments, even though the targeted genomic region was cut. These undesired on-target effects also had cellular consequences and significant implications for interpreting genome functionality. According to the study, the newly developed Xdrop-LRS approach showed the precise sequence content with sufficient length and resolution, which allowed the researchers to provide, for the first time, direct evidence that all of these genomic events can occur in combination on the same allele.

The researcher believes that their new findings and novel approach could be of great value for the national and international research communities, where CRISPR/Cas9 genome engineering has become the tool of choice for more and more researchers every day. The researchers also included guidelines for evaluating CRISPR/Cas9 deletion clones in their paper, which was published in the scientific journal Genome Research.

The study was a collaboration between SciLifeLab/KI and the University of York. Furthermore, one student (Linda Wedemann) was part of the SciLifeLab master program and did major contributions during her stay with us as a SciLifeLab summer student (funded by the SFO groups of all three universities on campus Solna).


Last updated: 2022-11-21

Content Responsible: Johan Inganni(