Here, we describe a protocol for a scaled-down version of a genomic DNA (gDNA)-fragmentation and tagmentation reaction using the Illumina Nextera XT DNA Library Preparation Kit. Using Staphylococcus aureus as an example, which has a genome size of ∼3 Mb, we show how 24 different samples can be pooled for a typical paired-end Illumina high-throughput sequencing run using the MiSeq Reagent V2 300-cycle kit, with which it is possible to sequence 5.1 Gb of DNA. As part of the protocol, a DNA size-selection method using a standard DNA agarose gel-extraction procedure and a final sample quality-control step using a Bioanalyzer are described.

Tools for site-directed mutagenesis of virulent bacteriophages (phages; viruses of bacteria) have traditionally lagged those for bacteria, hindering their study. CRISPR gene editing represents a new and highly efficient method for editing virulent phage genomes. Here, I describe methods for using CRISPR gene editing for site-directed mutagenesis of ICP1, a virulent phage of Vibrio cholerae The first section outlines methods of constructing a plasmid for CRISPR editing of an ICP1 gene. The second section outlines methods of transferring the plasmid to an editing-competent strain of V. cholerae The third section outlines methods of selecting for and storing the edited phage.

This protocol continues a series of methods for the construction of an in-frame gene deletion in Staphylococcus aureus strain RN4220. To this end, we describe in this protocol an allelic-exchange procedure for S. aureus We have previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-ΔtagO) can be constructed and isolated from Escherichia coli, then introduced into electrocompetent S. aureus cells by electroporation. This plasmid contains a temperature-sensitive origin of replication, a counterselectable marker (pheS* gene) and confers chloramphenicol resistance to S. aureus As a specific example, we present the construction of strain RN4220*ΔtagO from strain RN4220 carrying the pIMAY*-ΔtagO plasmid. The protocol can be easily adapted for the construction of other gene deletions and/or allelic-exchange plasmids.

Chromosomal mutations and targeted gene deletions and inactivations in Staphylococcus aureus are typically generated using the allelic exchange method. In recent years, however, more rapid methods have been developed, often using CRISPR-Cas9-based systems. Here, we describe recently developed CRISPR-Cas9-based plasmid systems for use in S. aureus, and discuss their use for targeted gene mutation and inactivation. First, we describe how a CRISPR-Cas9 counterselection strategy can be combined with a recombineering strategy to generate gene deletions in S. aureus We then introduce dead Cas9 (dCas9) and Cas9 nickase (nCas9) enzymes, and discuss how the nCas9 enzyme fused to different nucleoside deaminases can be used to introduce specific base changes in target genes. We then discuss how the nCas9-deaminase fusion enzymes can be used for targeted gene inactivation via the introduction of premature stop codons or by mutating the start codon. Together, these tools highlight the power and potential of CRISPR-Cas9-based methods for genome editing in S. aureus.

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.

Genome editing by site-directed mutagenesis is an important tool in biological research. CRISPR gene editing is the latest such tool developed, and one that is widely applicable to study organisms from all kingdoms of life. Here, I introduce a method for making site-directed, defined mutations in a virulent bacteriophage (a bacterial virus) using CRISPR gene editing. The ability to precisely edit the genomes of virulent phages will facilitate the study of their gene requirements for infection of host bacteria and advance our ability to engineer phages for use as therapeutic agents to combat bacterial infections. The protocol introduced here was developed as part of Cold Spring Harbor's Advanced Bacterial Genetics course.

This protocol is part of a series of methodologies for the construction of an in-frame gene deletion in Staphylococcus aureus strain RN4220. Having previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-ΔtagO) can be constructed and isolated from Escherichia coli, we now present details of the next steps in this method-the preparation of electrocompetent S. aureus cells and introduction of the tagO mutant plasmid DNA into the S. aureus cells by electroporation. Colonies containing the plasmid can then be selected on chloramphenicol plates at a low temperature permissive for plasmid replication.

In this protocol, we describe the basic steps for bacterial genome resequencing analysis using the QIAGEN CLC Genomics Workbench software. More specifically, we present how a reference genome sequence can be generated from Illumina reads of a wild-type reference bacterial strain and how this reference genome sequence can then be used to identify genomic alterations in mutant strains. As specific examples, Illumina reads from the Staphylococcus aureus RN4220 strain will be used to generate a consensus reference genome based on the publicly available S. aureus NCTC8325 genome sequence. The generated RN4220 consensus reference genome will subsequently be used to identify genomic mutations in an RN4220 mutant strain with increased oxacillin resistance (OxaR strain).