A5003857044- CRISPR and Genetic Engineering
- Bacterial Genetics and Biotechnology
- Insect symbiosis and bacterial influences
- RNA and protein synthesis mechanisms
- Wound Healing and Treatments
- Bone Tissue Engineering Materials
- RNA regulation and disease
- Antibiotic Resistance in Bacteria
- Antimicrobial agents and applications
- Advanced biosensing and bioanalysis techniques
Helmholtz Centre for Infection Research
2022-2024
Helmholtz Institute for RNA-based Infection Research
2022-2024
Abstract Bacterial genome editing commonly relies on chromosomal cleavage with Cas nucleases to counter-select against unedited cells. However, normally requires efficient recombination and high transformation efficiencies, which are unavailable in most strains. Here, we show that systematically attenuating DNA targeting activity enables RecA-mediated repair different bacteria, allowing drive editing. Attenuation can be achieved by altering the format or expression strength of guide (g)RNAs;...
Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA's presence Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram from diverse Cas12 nucleases, an dsDNA and subsequent collateral single-stranded DNA cleavage-all without RNA necessarily encoding protospacer-adjacent motif (PAM). After elucidating...
Abstract Capturing an individual cell’s transcriptional history is a challenge exacerbated by the functional heterogeneity of cellular communities. Here, we leverage reprogrammed tracrRNAs (Rptrs) to record selected transcripts as stored DNA edits in single living bacterial cells. Rptrs are designed base pair with sensed transcripts, converting them into guide RNAs. The RNAs then direct Cas9 editor target introduced target. extent editing can be read future sequencing. We use this approach,...
Abstract CRISPR-Cas systems can be utilized as programmable-spectrum antimicrobials to combat bacterial infections. However, how CRISPR nucleases perform across target sites and strains remains poorly explored. Here, we address this knowledge gap by systematically interrogating the use of using multidrug-resistant hypervirulent Klebsiella pneumoniae models. Comparing different Cas nucleases, DNA-targeting outperformed RNA-targeting based on tested targets. Focusing AsCas12a that exhibited...
ABSTRACT Bacterial genome editing commonly relies on chromosomal cleavage with Cas nucleases to counter-select against unedited cells. However, normally requires efficient recombination and high transformation efficiencies, which are unavailable in most strains. Here, we show that systematically attenuating DNA targeting activity enables RecA-mediated repair different bacteria, allowing drive editing. Attenuation can be achieved by altering the format or expression strength of guide (g)RNAs;...