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

• Published in: Scientific reports Vol. 6; no. 1; p. 23890
• Main Authors: Zhao, Yongping, Zhang, Congsheng, Liu, Wenwen, Gao, Wei, Liu, Changlin, Song, Gaoyuan, Li, Wen-Xue, Mao, Long, Chen, Beijiu, Xu, Yunbi, Li, Xinhai, Xie, Chuanxiao
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-04-2016; Nature Publishing Group
• Subjects: 13; 38/39; 42; 42/41; 42/70; 45; 45/22; 45/23; 45/77; 631/1647/1513/1967/3196; 631/61/338/552; Arabidopsis - genetics; Arabidopsis Proteins - genetics; CRISPR; CRISPR-Cas Systems; Crop improvement; Design; Gene Deletion; Gene Editing - methods; Gene Knockout Techniques; Genes, Plant; Genetic Vectors - genetics; Genome editing; Genomes; Homology; Humanities and Social Sciences; MicroRNAs - genetics; miRNA; multidisciplinary; Mutation; Plant breeding; Plants, Genetically Modified; Polymerase chain reaction; Recombination; RNA, Plant - genetics; Science; Science (multidisciplinary); Transformation, Genetic; Transgenic plants
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep23890

Precision 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.