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This repository contains the code used in the paper:

**A CRISPRi screen in E. coli reveals an unexpected sequence-specific toxicity of dCas9**
Lun Cui, Antoine Vigouroux, Francois Rousset, Hugo Varet, Varun Khanna & David Bikard


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In this study we designed a library of  guide RNAs targeting random positions
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along the genome of E. coli MG1655, with the simple requirement of a “NGG” PAM. The library 
contains an average of 19 targets per gene. A pool of guide RNAs obtained through on-chip 
oligo synthesis was cloned under the control of a constitutive promoter on plasmid psgRNA 
and electroporated in strain LC-E18 carrying the dCas9 gene under the control of a Ptet 
promoter in the chromosome. The pooled library of cells was then grown in rich medium 
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over 17 generations with anhydrotetracycline (aTc). The fold change in abundance (log2FC) of the guide RNA
was measured through deep sequencing of the library.
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The log2FC data for all guides in the screen can be found in the file screen_data.csv
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The analysis is divided in three jupyter notebooks:
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* Effect of dCas9 binding position and orientation.ipynb
> In this notebook we look at the effect of dCas9 binding position and orientation. In particular we perform an analysis of polar and reverse-polar effects.

* off-target analysis.ipynb
> In this notebook we investigate whether the unexpected fitness defect produced by some guides in the screen can be explained by off-target positions blocking the expression of essential or fitness genes.
  
* bad-seed analysis.ipynb
> In this notebook we use a machine learning approach to reveal how dCas9 can kill _E. coli_ when guided by some specific 5nt PAM-proximal sequences