title: Golden Gate Assembly of CRISPR gRNA expression array for simultaneously targeting multiple genes.
abstract: The engineered CRISPR/Cas9 technology has developed as the most efficient and broadly used genome editing tool. However, simultaneously targeting multiple genes (or genomic loci) in the same individual cells using CRISPR/Cas9 remain one technical challenge. In this article, we have developed a Golden Gate Assembly method for the generation of CRISPR gRNA expression arrays, thus enabling simultaneous gene targeting. Using this method, the generation of CRISPR gRNA expression array can be accomplished in 2 weeks, and contains up to 30 gRNA expression cassettes. We demonstrated in the study that simultaneously targeting 10 genomic loci or simultaneously inhibition of multiple endogenous genes could be achieved using the multiplexed gRNA expression array vector in human cells. The complete set of plasmids is available through the non-profit plasmid repository Addgene.
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, Building 1242, 8000, Aarhus C, Denmark.
authors.forename: Johan
authors.initials: J
authors.lastname: Vad-Nielsen
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, Building 1242, 8000, Aarhus C, Denmark.
authors.forename: Lin
authors.initials: L
authors.lastname: Lin
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, Building 1242, 8000, Aarhus C, Denmark.
authors.forename: Lars
authors.initials: L
authors.lastname: Bolund
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, Building 1242, 8000, Aarhus C, Denmark.
authors.forename: Anders Lade
authors.initials: AL
authors.lastname: Nielsen
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, Building 1242, 8000, Aarhus C, Denmark. alun@biomed.au.dk.
authors.forename: Yonglun
authors.initials: Y
authors.lastname: Luo
mesh_terms: D003001:Cloning, Molecular; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D005456:Fluorescent Dyes / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D056426:Genetic Loci; D006801:Humans; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: D005456:Fluorescent Dyes; D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: CRISPR; Cas9; Genome editing; Golden Gate Assembly; Simultaneous multiple gene inhibition; Simultaneous multiple gene knockout