Application of STAgR.

<p>(A) Colony PCR of a 6xSTAgR reaction using two different promoters as well as both, the canonical and the SAM loop gRNA scaffold. The gel shows a colony PCR of 22 bacterial colonies, of which seven showed the amplicon indicative of the full length STAgR reaction (2444bp). (B) Exemplary colony PCR of STAgR constructs with 0 to 8 gRNA expression cassettes. (C) A STAgR plasmid containing four gRNAs or a mixture of four single gRNA plasmids have been transfected into P19 Cells expressing dCas9-VPR. (D) After 7 days mRNA was extracted and transcript levels of target genes have been compared via qPCR. Error bars depict standard errors of the mean.</p

Functional validation of STAgR.

<p>(A) Colony PCR of a 4xSTAgR reaction (using a string sequence containing a hU6 promoter and a canonical gRNA scaffold). 24 bacterial colonies are shown, of which six present the amplicon size indicative of the full length reaction (1596 bp). Additionally marked are amplicon sizes indicative of two (823 bp) and single gRNAs (458 bp). (B) Quantification of cloning efficiencies from three different 4xSTAgR reactions (n = 130). (C) A schematic showing constructs used for functional validation of STAgR gRNAs. A gRNA targeting the GFP ORF was either delivered in a single gRNA expression vector or on each of four different positions in STAgR vectors. (D) Functional validation of STAgR vectors shown in Fig 2C. HeLa cells stably expressing d2GFP and Cas9 have been transfected with vectors depicted above. Flow cytometry indicates that STAgR constructs are similarly efficient in mutating the ORF of GFP compared to a single gRNA vector. (E) Colony PCR of a 4xSTAgR reaction using four different promoters and SAM loop scaffolds. 24 bacterial colonies are shown, of which seven colonies incorporated the amplicon size indicative of the full length reaction (2043 bp). Shorter amplicons are indicative of gRNA subsets, which vary in size, depending on the incorporated promoter.</p

The STAgR protocol.

<p>(A) An Overview over STAgR procedure. STAgR allows simple and fast generation of multiplexing vectors in one overnight reaction. STAgR is also highly customizable as diverse strings and vectors can be used to assemble expression cassettes with different promoters and gRNA scaffolds. (B) Sequences of overhang primers used for generation of STAgR vectors.</p