AsianScientist (Mar. 12, 2019) – A team of international scientists has developed a technique to evaluate the safety of genome-editing techniques. The research was published in Science.
CRISPR-Cas9 is a new generation of gene-editing tool that has been widely used. However, the risk of off-target effects in vivo, which could lead to diseases such as cancer, remains 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 from background noise. Whether CRISPR-Cas9 induces off-target effects has been controversial.
In the present study, researchers led by Professor Yang Hui of the Shanghai Institutes for Biological Sciences, China, in collaboration with colleagues at Stanford University, US, developed a method called genome-wide off-target analysis by two-cell embryo injection (GOTI) to identify off-target effects during gene editing.
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 was performed on the tdTomato+ and tdTomato– cells, respectively. Single nucleotide variations (SNV), insertions and deletions were then 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 attributed to genome-editing tools.
Using GOTI, the researchers first tested the CRISPR-Cas9 system, finding that it did not exhibit obvious 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.
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, said the researchers.
The article can be found at: Zuo et al. (2019) Cytosine Base Editor Generates Substantial Off-target Single-nucleotide Variants in Mouse Embryos.
———
Source: Chinese Academy of Sciences; Photo: Shutterstock.
Disclaimer: This article does not necessarily reflect the views of AsianScientist or its staff.
