Recently, researchers from the laboratories of Profs. GAO Caixia and LI Jiayang engineered five saturated targeted endogenous mutagenesis editors (STEMEs) and generated de novo mutations to facilitate directed evolution of plant genes.

 

Heredity and variation are the basis of organismic evolution. Random mutagenesis by physical (for example, ultraviolet) or chemical (for example, ethylmethane sulfonate) methods has long been applied to improve traits in plants, but is labor-intensive and time-consuming.

 

Although directed evolution is an effective strategy for obtaining desired genetic or protein variants, most approaches to library diversification involve error-prone PCR, DNA synthesis, recombination or mutator strains. In higher organisms, especially in plants, a target gene is usually transferred into a bacterial or yeast cell to generate the required diversity for selection, but once a target gene is not in situ, the functional consequences of changes may not be the same as in the native context. Moreover, most important agronomic traits could not be selected in bacterial or yeast.

 

Thus, as Prof. GAO Caixia said, “To establish powerful tools for directly inducing saturated targeted mutations and selection in plants will accelerate the development of agronomic traits and important functional genes.”

 

They fused cytidine deaminase with adenosine deaminase to obtain four saturated targeted endogenous mutagenesis editors (STEMEs). All four STEMEs produced simultaneous C>T and A>G conversions efficiently using only one single sgRNA. They also produced the fifth dual cytosine and adenine base editor STEME-NG to expand the targeting scope. With only 20 individual sgRNAs in rice protoplasts, STEME-NG could produce near-saturated mutagenesis for a 56-amino-acid portion of the rice acetyl-coenzyme A carboxylase (OsACC).

 

In a proof-of-concept experiment, they used STEMEs for directed evolution of ACC in rice plants. The regenerated rice seedlings were sprayed with haloxyfop as the selection pressure. They identified four novel (P1927F, W2125C, and S1866F) and one known (W2125C) amino acid substitutions of herbicide resistance. These mutations were found affecting the haloxyfop-binding pocket directly or indirectly based on the homology model of the CT domain of yeast ACC.

 

The establishment of STEME paves a way for directed evolution of plant endogenous genes in situ, which is important for molecular design breeding. Moreover, this STEME system might also be applicable beyond plants; for example, for screening drug resistancemutants, altering cis-elements on noncoding regions and correcting pathogenic SNVs in cell lines, yeast or animals.

 

This work, entitled “Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors” has been published in Nature Biotechnology (DOI: 10.1038/s41587-019-0393-7).

 

This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences, the National Natural Science Foundation of China, and the National Key Research and Development Program of China.