title: Rapid construction of multiple sgRNA vectors and knockout of the Arabidopsis IAA2 gene using the CRISPR/Cas9 genomic editing technology.
abstract: IAA2 is a member of the Aux/IAA auxin responsive gene family in Arabidopsis thaliana. No iaa2 mutant has been reported until now, thus hindering its further mechanistic investigations. The normal genomic editing technology of CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) uses only a single guide RNA (sgRNA) to target one site in a specific gene, and the gene knockout efficiency is not high. Instead, multiple sgRNAs can target multiple sites; therefore, the efficiency may be improved. In the present investigation, we used the golden-gate cloning strategy and two rounds of PCR reactions to combine three sgRNAs in the same entry vector. The final expression vector was obtained by LR reactions with the destination vector containing the Cas9 expression cassette. Four out of the six sgRNAs were effective, and we also obtained a lot of insertion and deletion mutations. Compared with one sgRNA approach, multiple sgRNAs displayed higher gene-knockout efficiency and produced more germ-line mutants. Thus, we established a more rapid and efficient method and generated five mutants for further studies of IAA2 functions.
authors.affiliation: College of Life Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Ding-yuan
authors.initials: DY
authors.lastname: Liu
authors.affiliation: College of Life Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Qiu
authors.affiliation: College of Life Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Xiao-hui
authors.initials: XH
authors.lastname: Ding
authors.affiliation: College of Life Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Miao-miao
authors.initials: M
authors.lastname: Li
authors.affiliation: College of Life Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Mu-yuan
authors.initials: MY
authors.lastname: Zhu
authors.affiliation: College of Life Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Jun-hui
authors.initials: JH
authors.lastname: Wang
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics*; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D000072669:Gene Editing / Q000379:methods; D055786:Gene Knockout Techniques / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D017393:RNA Editing / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D029681:Arabidopsis Proteins; C530977:IAA28 protein, Arabidopsis; D000094704:RNA, Guide, CRISPR-Cas Systems; D014157:Transcription Factors