Recently developed CRISPR-mediated base editors, which enable the generation of numerous nucleotide changes in target genomic regions, have been widely adopted for gene correction and generation of crop germplasms containing important gain-of-function genetic variations. However, to engineer target genes with unknown functional SNPs remains challenging. To address this issue, we present here a base-editing-mediated gene evolution (BEMGE) method, employing both Cas9n-based cytosine and adenine base editors as well as a single-guide RNA (sgRNA) library tiling the full-length coding region, for developing novel rice germplasms with mutations in any endogenous gene. To this end, OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium-mediated and particle-bombardment-mediated transformation. Four different types of amino acid substitutions in the evolved OsALS1, 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 bispyribac-sodium. Furthermore, the P171F substitution identified in a strong OsALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing with the corresponding base editor and sgRNA. Collectively, these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement.

最近开发的CRISPR介导的碱基编辑器能够在靶基因组区域中产生大量核苷酸改变，已被广泛用于基因校正和包含重要功能增益遗传变异的农作物种质的产生。然而，工程改造具有未知功能SNP的靶基因仍然具有挑战性。为了解决这个问题，我们在这里介绍一种碱基编辑介导的基因进化（BEMGE）方法，该方法同时使用基于Cas9n的胞嘧啶和腺嘌呤碱基编辑器，以及一个在全长编码区进行拼接的单向导RNA（sgRNA）文库，用于开发任何内源基因均具有突变的新型水稻种质。为此，OsALS1被人为在水稻细胞通过两个使用BEMGE演变农杆菌介导的和粒子轰击介导的转化。在进化的OsALS1中鉴定了四种不同类型的氨基酸取代，这些氨基酸取代来自水稻驯化过程中从未被自然或人为选择的两个位点，从而赋予了除草剂双嘧啶钠不同的耐受水平。此外，通过使用相应的碱基编辑器和sgRNA进行精确的碱基编辑，可将在强OsALS1等位基因中鉴定出的P171F替代快速引入商业水稻品种Nangeng 46。总体而言，这些数据表明BEMGE在创建用于作物改良的目标基因重要遗传变异中具有巨大潜力。