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Bikard Lab CRISPR Repression Plasmids

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The David Bikard Lab at the Institute Pasteur has deposited a collection of CRISPR arrays to modulate the expression levels of two targets independently.

The RNA-programmed DNA-binding protein dCas9 is expressed along a CRISPR array that contains 20 bp "addresses", where dCas9 is going to bind on the chromosome. When dCas9 binds to a gene, it blocks the progression of the RNA polymerase, thus strongly decreasing the expression level. Mismatches between the guide RNA and the target sequence lower the repression level. By controlling the number of mismatches we can obtain a range of expression levels.

This collection comprises 20 plasmids, each carrying two CRISPR guides. The first one binds to sfgfp with either 0, 10, 11, 14, or 20 matching nucleotides, the second binds to mcherry with either 0, 11, 18, or 20 matching nucleotides. Together, these plasmids allow to explore the whole range of expression levels for these two reporters simultaneously. By fusing sfGFP and/or mCherry to genes of interest, it is thus possible to study the effect of the amount and stoichiometry of enzymes.

The pLC97 plasmid allows for quick integration of the aTc-inducible pTet-dCas9 in the attachment site of the phage 186.

Detailed information about the repression system can be found in this article:

Vigouroux, A., Oldewurtel, E., Cui, L., Bikard, D., & Van Teeffelen, S. (2018). Tuning dCas9’s ability to block transcription enables robust, noiseless knockdown of bacterial genes. Molecular Systems Biology, 14(3):e7899. doi: 10.15252/msb.20177899. PMID: 29519933. bioRxiv preprint.

Detailed information about chromosomal integration of pLC97 can be found in this article:

St-Pierre, F., Cui, L., Priest, D. G., Endy, D., Dodd, I. B., & Shearwin, K. E. (2013). One-Step cloning and chromosomal integration of DNA. ACS Synthetic Biology, 2(9), 537–541. DOI: 10.1021/sb400021j

Panel A shows a schematic of the modified chromosome in E. coli containing the dCas9 and sfgfp and mCherry reporters, alongside a schematic of the CRISPR array in a plasmid containing the guide RNAs, CRISPR motifs, and tracerRNA sequences. Panel B is a graph with RFP fluorescence on the Y axis, GFP fluorescence on the X axis. See figure legend for details on the graph.
Figure 1: Multiplexing CRISPR knockdown to modulate expression of two genes without cross‐talk. (A) Schematic of the strain expressing two reporters and dCas9 under a P-tet promoter integrated in the chromosome at phage attachment sites. The levels of the two reporters, mCherry and sfgfp in this case, can be controlled using a plasmid‐borne CRISPR array coding for guide RNAs (diamonds) interspaced with CRISPR repeat motifs (squares), and also carrying the tracrRNA sequence (not shown). (B) Relative GFP and RFP concentration given relatively to the non‐targeting spacer measured by high‐throughput microscopy for a collection of 20 CRISPR plasmids. Each point represents a single cell, and each color represents the population obtained with one CRISPR plasmid. An overlaid meshwork connects the median values of the different populations. Image and caption from Vigouroux et al., 2018, under a CC BY 4.0 license.