Recently, Prof. LAI Liangxue from Guangzhou Institutes of Biomedicine and Health (GIBH) of the Chinese Academy of Sciences collaborating with Dr. ZOU Qingjian from Wuyi University, has developed a novel base editing system, TaC9-ABE, which is able to eliminate Cas9-dependent off-targets of adenine base editor (ABE). This work was published in Cell Discovery. 

Base editor enables single-nucleotide conversions without creating double-stranded DNA breaks. As an efficient and precise gene-editing tool, it can be used to generate gene-editing organisms and correct pathogenic point mutations for gene therapy.   

ABE can specifically convert adenine (A) to guanine (G) at the target sites. The canonical ABE contains a fusion protein composed of nCas9 and adenine deaminase and is guided by sgRNAs to create A-to-G conversion. However, sgRNAs tend to bind some sites with one or more mismatched nucleotides in genomes, thus resulting in off-target gene editing, which is the major safety concern in the generation of gene editing organisms and gene therapy for human diseases.  

In the TaC9-ABE editing system developed in this study, nCas9 is guided to the target site by sgRNA, while the adenine deaminase is guided by transcription activator-like effector (TALE). Only when nCas9 is guided to the target site close to the TALE target to open the DNA duplex and nick the targeted strand, TALE-guided adenine deaminase can convert A to G on the on-target single strand. If nCas9 is guided to a wrong site (mismatched off-target site) by sgRNA, base editing is not generated due to the absence of deaminase. Similarly, when deaminase is guided to the wrong site by TALE, the base editing will not happen due to the absence of single-strand DNA substrates. Thus, such base editing system can completely rule out the possibility of Cas9-dependent off-target.  

TaC9-ABE was confirmed to maintain the high base editing efficiency and to not lead to any detectable genomic off-target modification at off-target sites. By contrast, half of the Cas9-dependent off-targets were observed in the canonical adenine base editor, ABE7.10.   

“Our research solves the safety issue of single base editing technology in biomedical applications, providing a safer and more efficient tool for the treatment of human genetic diseases caused by point mutations,” Prof. LAI said.