CRISPR gives us the ability to precisely edit genes within cells, and the technique holds enormous potential for transforming the gene therapy space. However, concerns about CRISPR editing genes other than the ones it is targeted to have hindered its introduction into patient treatments. Known as off-target mutations, these unintended gene edits caused by CRISPR could cause cancer or other diseases.

To ensure that CRISPR can be safely used in patients, a greater understanding of both the frequency and impacts of off-target mutations will be required.

The CRISPR technique requires short sections of guide RNA to enable the enzyme Cas-9 to bind to its intended gene target. The researchers designed 58 gene editing experiments, each targeting a different gene in mice embryos. Each gene required 3-4 guide RNAs. The whole genomes of each mice were then sequenced to determine how many mutations, other than the targeted edit, had been induced by the CRISPR. To identify baseline mutation levels, the CRISPR-edited mice were compared to un-edited controls.

31 of the mice displayed no off-target mutations whilst 20 mice displayed an average of 2-3 off-target mutations. However, both
the treated and untreated mice lines displayed an average of 3,500 natural mutations. This demonstrates that the number of naturally occurring mutations far exceed the number of mutations introduced by CRISPR, and that off-target mutations are rare if effective guide RNA molecules are used.

The researchers next aim to investigate if different enzymes involved in DNA repair could reduce the risk of off target mutations. The findings can be applied in experimental design to test the safety of CRISPR gene therapies and aid the development of more precise guide RNAs.