An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design

dc.creatorYongping Zhao
dc.creatorCongsheng Zhang
dc.creatorWenwen Liu
dc.creatorWei Gao
dc.creatorChanglin Liu
dc.creatorGaoyuan, Song
dc.creatorWen-Xue Li
dc.creatorLong Mao
dc.creatorCheng, Beijiu
dc.creatorYunbi Xu
dc.creatorXinhai Li
dc.creatorChuanxiao Xie
dc.date2017-05-09T19:11:24Z
dc.date2017-05-09T19:11:24Z
dc.date2016
dc.date.accessioned2023-07-17T20:01:13Z
dc.date.available2023-07-17T20:01:13Z
dc.identifierhttp://hdl.handle.net/10883/18311
dc.identifier10.1038/srep23890
dc.identifier.urihttps://repositorioslatinoamericanos.uchile.cl/handle/2250/7510777
dc.descriptionPrecision DNA/gene replacement is a promising genome-editing tool that is highly desirable for molecular engineering and breeding by design. Although the CRISPR/Cas9 system works well as a tool for gene knockout in plants, gene replacement has rarely been reported. Towards this end, we first designed a combinatory dual-sgRNA/Cas9 vector (construct #1) that successfully deleted miRNA gene regions (MIR169a and MIR827a). The deletions were confirmed by PCR and subsequent sequencing, yielding deletion efficiencies of 20% and 24% on MIR169a and MIR827a loci, respectively. We designed a second structure (construct #2) that contains sites homologous to Arabidopsis TERMINAL FLOWER 1 (TFL1) for homology-directed repair (HDR) with regions corresponding to the two sgRNAs on the modified construct #1. The two constructs were co-transformed into Arabidopsis plants to provide both targeted deletion and donor repair for targeted gene replacement by HDR. Four of 500 stably transformed T0 transgenic plants (0.8%) contained replaced fragments. The presence of the expected recombination sites was further confirmed by sequencing. Therefore, we successfully established a gene deletion/replacement system in stably transformed plants that can potentially be utilized to introduce genes of interest for targeted crop improvement.
dc.formatPDF
dc.languageEnglish
dc.publisherNature Publishing Group
dc.relationhttps://www.nature.com/articles/srep23890#Sec23
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dc.rightsOpen Access
dc.sourceart. 23890
dc.source6
dc.sourceScientific Reports
dc.subjectAGRICULTURAL SCIENCES AND BIOTECHNOLOGY
dc.subjectGENES
dc.subjectPLANTS
dc.subjectDNA SEQUENCE
dc.subjectCHROMOSOME MANIPULATION
dc.titleAn alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design
dc.typeArticle
dc.coverageLondon


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