New Base Editing System Developed for Creating Saturated Mutant Populations

Recently, LAI Liangxue’s Lab from Guangzhou Institute of Biomedicine and Health (GIBH) of Chinese Academy of Sciences (CAS) has developed a new type of dual deaminase-mediated base editing system, the AGBE system. It can widely induce multiple editing patterns and serve as a flexible tool for establishing saturated mutant populations for the verification of functions and consequences of specific gene mutations. 

This study was published in Nucleic Acids Research on May 11, 2022. 

With high-throughput genome sequencing, previous investigations have detected a mass of single nucleotide variants (SNVs) associated with traits of crops and livestock or genetic diseases in humans. Identifying the relationship between these genetic variations and species traits and clinical phenotypes helps advance agricultural molecular breeding and precision medicine research. However, for limited gene editing technologies, it is a major challenge to understand the functions or consequences of most SNVs. 

In this study, the researchers built a dual deaminase-mediated AGBE system by fusing a glycosylase base editor (CGBE) with an adenine base editor (ABE) for saturated mutagenesis in a specific gene to discover the relationship between these genetic variations and the corresponding phenotypes in mammalian cells. They proved that the AGBE system was able to simultaneously introduce 4 types of base conversions (C-to-G, C-to-T, C-to-A and A-to-G) as well as indels at an expanded editing window. It was helpful for establishing saturated mutant populations due to its character of inducing various mutant patterns with a single sgRNA. Moreover, it did not significantly cause DNA or RNA off-target editing according to the results of whole genome sequencing and RNA sequencing, suggesting that it was an efficient and safe base editor. 

To verify AGBEs’ potential in screening functional mutations through saturated mutagenesis, the researchers employed miniAGBE-4, the optimal version of AGBE system, to install loss-of-function mutations in human diphtheria toxin receptor (hDTR) gene. The human cells with hDTR resistant mutation would survive and proliferate to form a population under the pressure of diphtheria toxin. Thus, 59269 types of base-edited alleles in total were generated by 20 sgRNAs after treatment of diphtheria toxin. Such results indicated that the AGBE system could be widely useful for generating saturated mutant populations for high-throughput screening of SNVs. 

This work was financially supported by the National Natural Science Foundation, the National Key Research and Development Program, the Science and Technology Planning Project of Guangdong Province, the Youth Innovation Promotion Association of CAS, and the Young Elite Scientist Sponsorship Program of China Association for Science and Technology.