Imperial College London
Alternate

Professor Angelika Gründling

Faculty of Medicine, Department of Infectious Disease

Professor of Molecular Microbiology
 
 
 
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Contact

 

+44 (0)20 7594 5256a.grundling Website

 
 
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Location

 

6.22Flowers buildingSouth Kensington Campus

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Summary

 

Publications

Citation

BibTex format

@article{Gründling:2023:10.1101/pdb.prot107925,
author = {Gründling, A and Ji, Q},
doi = {10.1101/pdb.prot107925},
journal = {Cold Spring Harb Protoc},
title = {Introduction of a CRISPR-nCas9 Gene-Targeting Plasmid into Staphylococcus aureus for Gene Disruption.},
url = {http://dx.doi.org/10.1101/pdb.prot107925},
year = {2023}
}
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RIS format (EndNote, RefMan)

TY  - JOUR
AB  - Methods for gene disruption are essential for functional genomics, and there are multiple approaches for altering gene function in bacteria. One of these methods involves introducing a premature stop codon in a gene of interest, which can be achieved by using the CRISPR-nCas9-cytidine deaminase system. The approach involves the mutation of editable cytidines to thymidines, with the goal of generating a novel stop codon that ultimately results in a nonfunctional gene product. The workflow involves two major sections, one for the identification of editable cytidines, the design of the targeting spacer oligonucleotides for introduction into the CRISPR-nCas9 cytidine deaminase plasmid, and the construction of the gene-targeting CRISPR-nCas9 cytosine deaminase plasmids, and one for the actual introduction of the mutation in the species of interest. Here, we describe the steps for the second part. Specifically, we describe (1) how to introduce the gene-targeting pnCasSA-BEC plasmid into Staphylococcus aureus, (2) how the gene inactivation in S. aureus can be confirmed by PCR and sequencing, and (3) how, following successful gene inactivation, the strain can be cured of the pnCasSA-BEC plasmid. To better illustrate the method, and as specific example, two different geh gene-inactivation mutations are generated here in S. aureus RN4220. The protocol, however, can easily be adapted to generate other gene-inactivating mutations.
AU  - Gründling,A
AU  - Ji,Q
DO  - 10.1101/pdb.prot107925
PY  - 2023///
TI  - Introduction of a CRISPR-nCas9 Gene-Targeting Plasmid into Staphylococcus aureus for Gene Disruption.
T2  - Cold Spring Harb Protoc
UR  - http://dx.doi.org/10.1101/pdb.prot107925
UR  - https://www.ncbi.nlm.nih.gov/pubmed/37117019
ER  -
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