Three Novel Alleles of OsGS1 Developed by Base-Editing-mediated Artificial Evolution Confer Glufosinate Tolerance in Rice

Only few glufosinate-tolerant genes, such as phosphinothricin acetyltransferase（PAT） and bialaphos resistance（bar） identified from Streptomyces, are currently available for developing genetically modified rice in agricultural application. Following the rapid development of genome editing technology, generation of novel glufosinate-tolerant gene resources through artificial evolution of endogenous genes is more promising and highly desirable in rice molecular breeding program. In this study, the endogenous Glutamine synthetase1（OsGS1） was artificially evolved by base-editing-mediated gene evolution（BEMGE） in rice cells to create novel alleles conferring glufosinate tolerance in rice germplasms. Two novel glufosinate-tolerant OsGS1 alleles（OsGS1-AVPS and OsGS1-+AF） and one reported tolerant allele（OsGS1-SGTA） were successfully identified from approximately 4200 independent hygromycin-tolerant calli. Germination assays and spray tests revealed that these three OsGS1 alleles confer glufosinate tolerance in rice. Furthermore, OsGS1-AVPS and OsGS1-SGTA were quickly deployed into the elite rice cultivar Nangeng 46 through precise base editing. Overall, our results demonstrate the feasibility of developing glufosinate-tolerant rice by editing an endogenous rice gene in molecular breeding programs.