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| Status |
Public on Feb 05, 2024 |
| Title |
Enhanced genome-wide knockout screens in bacteria with CRISPR base editors |
| Organism |
Escherichia coli |
| Experiment type |
Other
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| Summary |
Genome-wide knockout or knockdown screens have become powerful tools for the investigation of genotype-to-phenotype relationships. In bacteria, these screens commonly rely on transcriptional repression by dCas9, gene knockouts through Cas9 editing or random transposon mutagenesis, but depending on the technique, suffer from incomplete gene silencing, low editing efficiencies or they require massive library sizes. Here, we take a distinct approach with base editing to introduce premature stop codons or mutate start codons in Escherichia coli using a ScCas9 nickase derived base editor (ScBE3) that exhibits flexible PAM recognition. We then derive guide design rules by applying machine learning to a gene essentiality screen conducted in E. coli. For further improvement, we combined base-editing with Cas9-induced cleavage of the unedited cell fraction. The efficiency of this dual system was validated through a screen of conditionally essential E. coli genes. This improved setup that decouples the gene editing from the screening leads to more efficient guide depletion and confirmed previously published conditionally essential genes. Overall, base editing represents a useful tool for genome-wide knockout screens in bacteria and will eventually enable genome-wide knockout screens in a broader range of bacterial species to study their diverse genetics.
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| Overall design |
Illumina sequencing reads from the genome-wide essentiality screen in E. coli; samples include the extracted library DNA from experiments with two base editor variants (sample label “s” and “e”) and 4 time points each (t1-4). The experiment was conducted in duplicate (e.g. e1 and e2). Illumina sequencing reads from the dual-sgRNA screen in E. coli; sample include the initial library, the extracted library after culturing in LB medium, after the base editing step, after the killing step and after screening in MOPS and M9 minimal media. Except for the initial library reads, all sample reads are provided in duplicates.
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| Contributor(s) |
Gawlitt S, Collins SP, Yu Y, Blackman S, Barquist L, Beisel CL |
| Citation(s) |
38499498 |
| |
| Submission date |
Feb 15, 2023 |
| Last update date |
May 10, 2024 |
| Contact name |
Chase Beisel |
| E-mail(s) |
chase.beisel@helmholtz-hiri.de
|
| Organization name |
Helmholtz Institute for RNA-based Infection Research
|
| Street address |
Josef-Schneider-Str. 2 / Bau D15
|
| City |
Wuerzburg |
| ZIP/Postal code |
97080 |
| Country |
Germany |
| |
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| Platforms (2) |
| GPL21222 |
Illumina NextSeq 500 (Escherichia coli) |
| GPL25368 |
Illumina NovaSeq 6000 (Escherichia coli) |
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| Samples (27)
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| Relations |
| BioProject |
PRJNA935297 |
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