Sequencing results of the rBE14-induced OsWRKY45 mutations in T0 transgenic rice callus lines. (I) Summary of nucleotide changes in the editing window of the endogenous OsWRKY45 gene in T0 transgenic rice callus lines. (J) Summary of base editing efficiencies of rBE14 with different DNA contexts at five genomic sites. (K) Visualizing GFP in transgenic rice callus under a handheld UV lamp and confocal microscope. For (B), (E), (G), (H), and (I), The PAM sequence, the candidate bases in the putative editing window, the detected nucleotide changes/the corresponding amino acids and the sgRNA sequence are highlighted in green, red, blue and bold, respectively. For (C) and (F), the nucleotide changes are underlined in the sequencing chromatograms.
The target site of pi-d2 gene in rice. (D) Representative Sanger sequencing chromatogram of the rBE5-edited pi-d2 alleles with the target G changed in T0 transgenic rice line #3. (E) Efficiency of rBE5-versus rBE3-mediated gene correction of pi-d2 in T0 transgenic rice lines. (F) The target site of OsFLS2 gene in rice. (G) Representative Sanger sequencing chromatogram of the rBE5-edited OsFLS2 alleles with anticipated C > A conversion in T0 transgenic rice line #21. (H) Efficiency of the rBE5 system in base editing OsFLS2 in T0 transgenic rice lines. (I) Sequencing results of the rBE5-induced OsFLS2 mutations in T0 transgenic rice lines. (J) Construct of the rBE9 system used to induce nucleotide changes in rice transgenic plants. (K) Efficiencies and ratios of allelic mutations caused by the rBE3 and rBE9 systems. (L) Base editing efficiencies of the rBE3 and rBE9 systems at the target C in different sequence context. For (C, F, and I), the PAM sequences, the putative target bases in the activity window, and the detected nucleotide/corresponding amino acids are highlighted in green, red, and blue, respectively. For (D and G), nucleotide mutations are underlined in the sequencing chromatograms.
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.
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