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Series GSE237136 Query DataSets for GSE237136
Status Public on Jul 17, 2023
Title Systematic interrogation of CRISPR-based antimicrobials in Klebsiella pneumoniae reveals nuclease-, guide-, and strain-specific factors influencing antimicrobial activity
Organisms Escherichia coli; Klebsiella pneumoniae
Experiment type Expression profiling by high throughput sequencing
Summary CRISPR-Cas systems can be utilized as programmable-spectrum antimicrobials to combat bacterial infections. However, how CRISPR nucleases perform as antimicrobials across target sites and strains remains poorly explored. Here, we address this knowledge gap by systematically interrogating the use of CRISPR antimicrobials against multidrug-resistant and hypervirulent strains of Klebsiella pneumoniae. Comparing different Cas nucleases, we found that AsCas12a exhibited robust targeting across different strains. The elucidated modes of escape from this nuclease varied widely, restraining opportunities to enhance killing. We also encountered individual guide RNAs yielding different extents of clearance across strains. The differences were attributed to improper RNA folding, leading to inefficient DNA cleavage and subsequent repair via homologous recombination. To explore features that could improve targeting across strains, we performed a genome-wide screen in different K. pneumoniae strains that yielded guide design rules and trained an algorithm for predicting guide efficiency. Finally, we showed that Cas12a antimicrobials can be exploited to eliminate K. pneumoniae when encoded in phagemids delivered by T7-like phages. Altogether, our results highlight the importance of evaluating antimicrobial activity of CRISPR antimicrobials across relevant strains and define critical parameters for efficient CRISPR-based targeting.
 
Overall design A library (11,900 targets and 100 non-targeting guides) was designed to target a non-redundant collection of complete genomes of Klebsiella pneumoniae, by randomly choosing targets with a proper TTTV PAM shared by all the genomes in the collection. The library was prepared in E. coli MG1655, transfered to E. coli MFDpir, and delivered by conjugation to 3 K. pneumoniae strains (NTUH-K2044, KPPR1 and SB5442). Samples were harvested after induction with 1 nM anhydrotetracycline (aTc) at time zero, 3 hours and 16 hours. Finally, plasmids were extracted from 10 mL of these cultures and the donor strain culture. The samples were prepared for amplicon sequencing and sequencing was then performed with Illumina sequencing primers using a NextSeq 500 benchtop sequencer
 
Contributor(s) Miele S, Bikard D, Vialetto E, Beisel C
Citation(s) 38661215
Submission date Jul 12, 2023
Last update date Jun 28, 2024
Contact name David Bikard
E-mail(s) dbikard@gmail.com
Organization name Institut Pasteur
Street address 28 rue du Dr Roux
City Paris
ZIP/Postal code 75015
Country France
 
Platforms (2)
GPL21222 Illumina NextSeq 500 (Escherichia coli)
GPL25165 Illumina NextSeq 500 (Klebsiella pneumoniae)
Samples (20)
GSM7595897 donor library, biol rep 1, E. coli MDFpir
GSM7595898 donor library, biol rep 2, E. coli MDFpir
GSM7595899 time 0, biol rep 1, K. pneumoniae strain SB5442
Relations
BioProject PRJNA994043

Download family Format
SOFT formatted family file(s) SOFTHelp
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Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE237136_RAW.tar 2.6 Mb (http)(custom) TAR (of TSV)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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