Chinese and international scientists have developed a new technique to evaluate the safety of genome-editing tools – a method that could become the industry standard. The research was published in Science on March 1. 

The technique – known as “GOTI,” for Genome-wide Off-target analysis by Two-cell embryo Injection – was developed by researchers from the Institute of Neuroscience (ION) of the Chinese Academy of Sciences (CAS), the CAS-MPG Partner Institute for Computational Biology of the Shanghai Institute of Nutrition and Health of CAS, Stanford University and the Agricultural Genome Institute at Shenzhen. 

The researchers used GOTI to evaluate genome-wide off-target effects induced by genome-editing tools including CRISPR/Cas9 and base editors. They found that cytosine base editors induced substantial off-target single-nucleotide variants (SNVs). They also showed that GOTI significantly improved the sensitivity of off-target detection in the absence of prediction and could detect randomly generated off-target variants. 

CRISPR/Cas9 is a new generation of gene-editing tool that has been widely used. However, the risk of off-target effects, such as cancer, has been a serious concern.  

A variety of off-target detection schemes have been developed, with most relying on the prediction of off-target sites based on sequence similarity or in vitro amplification. However, the latter process may introduce a large amount of noise, thus making it difficult to separate off-target signals – especially single-nucleotide variations – from background noise. Whether CRISPR/Cas9 induces off-target effects has been controversial. 

YANG Hui’s group from ION, along with the other collaborators, developed GOTI as a precise off-target detection method independent of computational prediction and with a high signal-to-noise ratio. The exquisiteness of this study is the use of mouse embryos in the experiment. 

Using a mouse embryo at the two-cell stage, the researchers edited and labeled one blastomere with a red fluorescent protein (tdTomato), leaving the other blastomere unedited. They then sorted the progeny cells of the edited and non-edited blastomeres by FACS based on tdTomato expression at embryonic day 14.5 (E14.5). 

Whole genome sequencing (WGS) was performed on the tdTomato+ and tdTomato- cells, respectively. SNVs and indels were identified by the overlap of three algorithms in the tdTomato+ sample, with the tdTomato- sample from the same embryo as the reference. This method made it possible to avoid the noise problem caused by in vitro amplification. 

Moreover, since the experimental group and the control group were both from the same embryo, the genetic background was completely identical. Therefore, the difference between the two cell populations was assumed to be caused by genome-editing tools.  

Using GOTI, the researchers first tested the CRISPR/Cas9 system; they found that it did not have obvious off-target effects. This result should quiet the long-standing controversy about CRISPR/Cas9 off-target effects. 

The researchers then tested another CRISPR/Cas9-derived technology, BE3. Previous studies had reported that BE3 introduced point mutations, but no significant off-target problems. However, the researchers discovered that BE3 generated substantial off-target SNVs that were not predicted by traditional off-target prediction methods. In addition, some of the off-target sites were found to appear on oncogene and tumor-suppressor genes. 

Using the classic version of BE3 for clinical application is currently a matter of great concern. Based on off-target detection by GOTI, the researchers found that some of the BE3-related gene editing tools produce unpredictable off-target risks. This finding encourages the world to re-examine the risks of such emerging technologies. 

In summary, this study established a gene-editing off-target detection method with higher precision, breadth and accuracy than previous methods. GOTI can be applied to develop a new generation of genome-editing tools with higher accuracy and safety, thus establishing a new industry standard. 

This study was sponsored by the R&D Program of China, the CAS Strategic Priority Research Program, the National Natural Science Foundation of China, the Shanghai Municipal Science and Technology Major Project Program and an NIH P01 Center grant.