基因靶向(GT)实现精确的基因组修饰-例如,引入碱基置换-使用供体DNA作为模板。结合用于选择GT细胞的选择标记的清洁切除,GT有望成为标准,一般适用,基地编辑系统。以前,我们证明了通过piggyBac转座子从水稻的GT修饰基因座中切除标记。然而,piggyBac介导的标记切除具有仅识别序列TTAA的局限性。最近,我们提出了一种新颖的通用精确基因组编辑系统,该系统由GT和随后的单链退火(SSA)介导的标记切除组成,其中有,原则上,没有靶序列的限制。在这项研究中,我们将碱基替换引入了OsCly1基因的microRNAmiR172靶位点,OsCly1基因是大麦Cleistogamy1基因的直系同源物,该基因与cleistogamy1开花有关。为了确保有效的SSA,GT载体在选择标记的两端具有1.2kb的重叠序列。使用具有重叠序列的载体的正-负选择介导的GT的频率与使用没有重叠序列的piggyBac介导的标记切除的载体获得的频率相当。在T0代中,SSA介导的标记切除的频率为〜40%。这种频率被认为足以产生无标记细胞,虽然它低于piggyBac介导的标记切除,接近100%。迄今为止,使用碱基编辑器和基于CRISPR/Cas9的主编辑系统在靶基因的不连续多个碱基中引入精确替换是相当困难的。这里,使用GT和我们的SSA介导的标记切除系统,我们不仅成功地对OsCly1基因的miR172靶位点中的单个碱基,而且成功地对人工不连续的多个碱基进行了精确的碱基置换。使用这种精确的基因组编辑系统对靶基因中的miRNA靶位点进行精确的碱基替换将是生产具有改善性状的有价值作物的有力工具。
Gene targeting (GT) enables precise genome modification-e.g., the introduction of base substitutions-using donor DNA as a template. Combined with clean excision of the selection marker used to select GT cells, GT is expected to become a standard, generally applicable, base editing system. Previously, we demonstrated marker excision via a piggyBac transposon from GT-modified loci in rice. However, piggyBac-mediated marker excision has the limitation that it recognizes only the sequence TTAA. Recently, we proposed a novel and universal precise genome editing system consisting of GT with subsequent single-strand annealing (SSA)-mediated marker excision, which has, in principle, no limitation of target sequences. In this study, we introduced base substitutions into the microRNA miR172 target site of the OsCly1 gene-an ortholog of the barley Cleistogamy1 gene involved in cleistogamous flowering. To ensure efficient SSA, the GT vector harbors 1.2-kb overlapped sequences at both ends of a selection marker. The frequency of positive-negative selection-mediated GT using the vector with overlapped sequences was comparable with that achieved using vectors for piggyBac-mediated marker excision without overlapped sequences, with the frequency of SSA-mediated marker excision calculated as ~40% in the T0 generation. This frequency is thought to be adequate to produce marker-free cells, although it is lower than that achieved with piggyBac-mediated marker excision, which approaches 100%. To date, introduction of precise substitutions in discontinuous multiple bases of a targeted gene using base editors and the prime editing system based on CRISPR/Cas9 has been quite difficult. Here, using GT and our SSA-mediated marker excision system, we succeeded in the precise base substitution not only of single bases but also of artificial discontinuous multiple bases in the miR172 target site of the OsCly1 gene. Precise base substitution of miRNA target sites in target genes using this precise genome editing system will be a powerful tool in the production of valuable crops with improved traits.