On-target activity predictions enable improved CRISPR–dCas9 screens in bacteria

Abstract

The ability to block gene expression in bacteria with the catalytically inactive mutant of Cas9, known as dCas9, is quickly becoming a standard methodology to probe gene function, perform high-throughput screens, and engineer cells for desired purposes. Yet, we still lack a good understanding of the design rules that determine on-target activity for dCas9. Taking advantage of high-throughput screening data, we fit a model to predict the ability of dCas9 to block the RNA polymerase based on the target sequence, and validate its performance on independently generated datasets. We further design a novel genome wide guide RNA library for E. coli MG1655, EcoWG1, using our model to choose guides with high activity while avoiding guides which might be toxic or have off-target effects. A screen performed using the EcoWG1 library during growth in rich medium improved upon previously published screens, demonstrating that very good performances can be attained using only a small number of well designed guides. Being able to design effective, smaller libraries will help make CRISPRi screens even easier to perform and more cost-effective. Our model and materials are available to the community through crispr.pasteur.fr and Addgene.

The impact of genetic diversity on gene essentiality within the E. coli species

“…We then selected 3-4 sgRNAs per gene following rules designed to favor targets that are conserved across strains while minimizing off-target activity and avoiding toxic seed sequences 33 ( see Methods ). We also used a model of dCas9 on-target activity to select the most active guides 39 . Our library also includes guides targeting rRNAs, tRNAs and widespread ncRNAs.…”

“…We used a recent model 39 which predicts the repression efficiency of sgRNAs based on fitness data obtained in a previous CRISPRi screen 33 . For each gene, the predicted sgRNA activity was normalized from 0 (highest activity) to 1 (lowest activity) and was then used as a 2 nd score.…”

Outer membrane lipoprotein DolP interacts with the BAM complex and promotes fitness during envelope stress response

“…Strain LC-E75 ( dolP + ) and its Δ dolP derivative were transformed with the EcoWG1 library which contains 5 guides per gene as previously described 39 . After culturing pooled transformant cells in LB at 37°C to early exponential phase (optical density at 600 nm [OD 600 ] = 0.2), a sample was withdrawn for plasmid isolation (t start ).…”

CRISPR interference strategies for studies in essential gene function and live cell fluorescent imaging of DNA elements in Escherichia coli

“…Moreover, the ability of CRISPRi to incrementally decrease transcription of targeted genes was recognized as a novel approach to better understand the role of essential bacterial genes in cell structure and function. (129,179).…”

“…Fortunately, more accurate predictions of the efficiency of guide RNA sequences for repression, with minimal off-targeting while avoiding bad seed sequences, can now be made for E. coli (180,182,195). For example, using machine learning to analyze genome-scale CRISPRi studies, the Bikard laboratory group has built a valuable on-line tool that allows the user to browse the targeting efficiency of the entire E. coli K-12 genome for individual guide RNAs, as well as provides an on-target activity prediction tool (https://crispr-browser.pasteur.cloud) (179).…”