Three recent studies suggest using the CRISPR-Cas9 gene editing tool to make changes to the DNA of human embryos results in large unwanted changes to the DNA near the target site of the CRISPR edit. Previous studies have revealed off-target edits could be made at locations far away from the targeted site, which are relatively easy to detect. The latest studies have shown that major changes occur close to the target site, which are difficult to detect using standard assessments. The latest research suggests the risk of using CRISPR-Cas9 may be much greater than previously thought.
The CRISPR-Cas9 system consists of a guide RNA which is programed to direct the Cas9 enzyme component to the correct section of DNA where it performs a cut of both strands of DNA. The DNA repair mechanisms of the cell then rejoin the DNA. While this technique can be used to remove a defective gene, errors can be introduced and some rearrangement of the DNA can occur and sections of the chromosome can be lost. Previous studies on mouse embryos have shown this to be the case, but it was not clear whether similar errors would be introduced in human embryos.
The first studies to be conducted on human embryos using CRISPR-Cas9 took place in 2015. Further studies have been conducted, but there are strict regulations that restrict the use of CRISPR-Cas9 on human embryos. Currently little is known about the processes involved in repairing cuts to the DNA in embryos. Knowledge of those processes is essential before CRISPR-Cas9 could be considered for use to edit out genetic defects in embryos.
One of the three studies was conducted at the Francis Crick Institute in London. CRISPR-Cas9 was used to create mutations in the POU5F1 gene, which plays an important role in embryonic development. The researchers used CRISPR-Cas9 on 18 embryos, and 22% were found to have large, unwanted changes to the DNA in areas surrounding the POU5F1 gene. In some cases, large sections of DNA consisting of several thousand bases had been deleted and there were multiple DNA rearrangements.
A second study was conducted by researchers at Columbia University. In this study, the embryos were created using sperm carrying a genetic defect that caused blindness through a mutation in the EYS gene. The researchers used CRISPR-Cas9 to correct the mutation. Half of the embryos lost large sections of the chromosome and, in some cases, the embryos lost the entire chromosome on which the EYS gene was located.
A third study conducted by researchers at Oregon Health & Science University used embryos created with sperm that caused a heart condition. The researchers also found that following editing with CRISPR-Cas9, large sections of the chromosome were lost. Pre-prints of each of the studies have been published at bioRxiv, although they have yet to go through the peer review process.
Most studies involving CRISPR-Cas9 look for unwanted, off-target edits of the DNA rather than the deletion of large sections of DNA or rearrangements, so these unwanted changes are often missed. These three studies show that the unwanted changes are not anomalies; they are common and must therefore be taken seriously.
The researchers have proposed several mechanisms that may be involved, but there is no consensus. Reproductive biologist, Shoukhrat Mitalipov, who led the study at Oregon Health & Science University, suggested gene conversion may have occurred. Gene conversion is where the repair mechanism copies a gene from another chromosome when making the repair to the cut DNA, but stem-cell biologist, Dieter Egli, who led the Columbia University study, searched for gene conversion and failed to find any evidence that it had occurred.
Further information on the findings of the three studies can be found in the journal Nature. DOI: 10.1038/d41586-020-01906-4