A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

Abstract

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic engineering became applicable to a large range of systems. Moreover, many transcriptional and epigenomic engineering approaches are now generally feasible for the first time. One reason for the broad applicability of CRISPR lies in its bipartite nature. Small gRNAs determine the genomic targets of the complex, variants of the protein Cas9, and the local molecular consequences. However, many CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into individual cells. Here, we present a customizable protocol for string assembly gRNA cloning (STAgR), a method that allows the simple, fast and efficient generation of multiplexed gRNA expression vectors in a single cloning step. STAgR is cost-effective, since (in this protocol) the individual targeting sequences are introduced by short overhang primers while the long DNA templates of the gRNA expression cassettes can be re-used multiple times. Moreover, STAgR allows single step incorporation of a large number of gRNAs, as well as combinations of different gRNA variants, vectors and promoters.

CRISPR-Mediated Induction of Neuron-Enriched Mitochondrial Proteins Boosts Direct Glia-to-Neuron Conversion

“…Quantitative RT-PCR from cells isolated by fluorescence-activated cell sorting (FACS) 48 h after transfection of the dCas9-VPR coding plasmid ( Breunig et al., 2018b ) and non-targeting control gRNAs or gRNAs designed to target the promoter region of the above candidates showed different levels of induction ( Figure 3 C; Arg2 , ∼94-fold; Acot7 , Prdx2 , Sod1 , Slc25a22 , Dnm3 , Mgst1 , and Prdx6 , ∼ 5-fold; Gls , Mgst3 , and Pgam5 , ∼2-fold). Multiple gRNAs targeting different genes (e.g., Arg2+Gls) did not alter the induction levels of their specific targets, and no significant induction was detectable for six putative off targets of each gRNA ( Figure S4 C).…”

“…For generation of STAgR cloning fragments, we followed previously published protocols (Breunig et al, 2018a(Breunig et al, , 2018bGibson, 2011). In particular, we generated individual cloning fragments for Gibson assembly by PCRs on 10 ng of vector templates (STAgR_Neo, STAGR_gRNAScaffold_hU6, STAGR_gRNAScaffold_hH1, STAGR_gRNAScaffold_h7SK and/or STAGR_gRNAScaffold_mU6).…”