Base Editing Mediated Gene Evolution

Genetic diversity is the basis of crop germplasm improvement,and many important agronomic traits of crops are determined by the single nucleotide polymorphism in the genome.However,how to use these variation information for rapid and accurate crop genetic improvement and solve the shortage of existing excellent variation resources have always been the focus and difficulty of agricultural scientific research.Recently CRISPR-mediated base editors,which achieve specific base substitutions in plant and animal genomes,have been widely adopted for gene correction and generation of crop germplasms containing important gain-of-function genetic variations.However,the specific PAM requirement for Cas9 recognition strongly restricts the targeting range of the CRISPR/Sp Cas9-mediated base editors because the targeted point mutation needs the availability of a PAM appropriately positioned and suitable base editing window.Extending the recognition range of Cas9,especially base editors,is an important direction for the optimization and improvement of the CRISPR/Cas9 system at present.Moreover,to engineer target genes with unknown functional SNPs and achieve directed evolution of proteins in planta remain challenging.In this study,the efficiency of both Sc Cas9 variant Sc++ toward four NNG PAMs and their application in both cytosine and adenine base editing was investigated in detail using transgenic rice to broaden the editing scope of CRISPR toolbox in rice.At the same time,we present here the base-editing-mediated gene evolution(BEMGE)method,a novel crop-breeding procedure that efficiently induces numerous nucleotide mutations in any target rice endogenous gene through the use of cytidine and adenosine base editors as well as a tiled sg RNA library,to identify novel alleles of different strength identify novel alleles of interested traits.The following results were achieved:Firstly,the performance of a Sc Cas9 variant named Sc++ at different NNG PAM sites in the rice genome was investigated in this study.Sc++ harbors a T1227 K mutation and the substitution of a positively charged loop(residues 367–376),derived from Streptococcus gordonii Cas9 and Streptococcus anginosus Cas9 respectively.The results showed that Sc++ nuclease improved targeted base editing in transgenic rice plants,and editing events occurred at some target sites not recognized by Sc Cas9.Meanwhile Sc++ overcame the site-dependence of Sc Cas9 to an extent,indicating that Sc++achieved broader genome editing.Then,Sc++-engineered cytosine base editor r BE71 and adenine base editor r BE73 b by fusing h AID*Δ and Tad A9 were then developed and achieved efficiently C>T and A>G substitutions by recognizing NAG,NGG and NCG PAM in the rice genome,respectively.Using the high-efficiency adenine base editor r BE73 b,we generated new Os GS1 alleles suitable for screening of rice germplasm for potential herbicide resistance in the future.The CRISPR/Sc++ system expands the genome-editing toolkit for rice.Natural and spontaneous mutation are the natural way by which evolution occurs.Based on the various efficient rice base editors previously established,we proposed a base-editing-mediated gene evolution(BEMGE)method to mimic the natural evolution process in planta at an accelerated pace,employing both Cas9n-based cytosine base editor r BE9 and adenine base editor r BE14 as well as a single-guide RNA(sg RNA)library tiling the full-length coding region,for developing novel rice germplasms with mutations in interested endogenous gene.To this end,herbicide target gene Os ALS1 was artificially evolved in rice cells using BEMGE,we designed 63 sg RNAs(NGG PAM)tiling the coding region of Os ALS1,and introduced the r BE/sg RNA plasmid library into rice cells through both Agrobacterium-mediated and particle-bombardment-mediated transformation.We first cultured transgenic calli with Os ALS1 on callus induction medium without bispyribac-sodium(BS)selection.The results of amplicon deep sequencing and Sanger sequencing showed that BEMGE could cover all11 sg RNAs and SNVs were enriched around the region targeted.Combining all the data,we conclude that BEMGE using a sg RNA library is capable of inducing rapid artificial evolution of the endogenous Os ALS1 gene in rice cells.Next,approximately 3600 independent hygromycin-resistant calli,generated through both Agrobacterium-mediated and particle-bombardment-mediated transformation with the six r BE/sg RNA pools,were subcultured on MSD plates containing 0.4 μM BS for screening evolved Os ALS1 variants which conferring BS tolerance.Four different types of amino acid substitutions(P171F,P171 SL,P171S,and R190H)in the evolved Os ALS1,derived from two sites that have never been targeted by natural or human selection during rice domestication,were identified,conferring varying levels of tolerance to the herbicide BS.Furthermore,the P171 F substitution identified in a strong Os ALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing with the corresponding base editor and sg RNA.Collectively,these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement.