CRISPR/Cas9 Editing of the Bacillus subtilis Genome

Abstract

A fundamental procedure for most modern biologists is the genetic manipulation of the organism under study. Although many different methods for editing bacterial genomes have been used in laboratories for decades, the adaptation of CRISPR/Cas9 technology to bacterial genetics has allowed researchers to manipulate bacterial genomes with unparalleled facility. CRISPR/Cas9 has allowed for genome edits to be more precise, while also increasing the efficiency of transferring mutations into a variety of genetic backgrounds. As a result, the advantages are realized in tractable organisms and organisms that have been refractory to genetic manipulation. Here, we describe our method for editing the genome of the bacterium Bacillus subtilis. Our method is highly efficient, resulting in precise, markerless mutations. Further, after generating the editing plasmid, the mutation can be quickly introduced into several genetic backgrounds, greatly increasing the speed with which genetic analyses may be performed.

The nucleotide pGpp acts as a third alarmone in Bacillus, with functions distinct from those of (p)ppGpp

“…Δ nahA markerless deletion of Bacillus subtilis was constructed by CRISPR/Cas9 editing method 49 : CRISPR editing plasmid pJW737 was constructed by Golden Gate assembly method (New England BioLabs) using pPB41 backbone, guide RNA template, and DNA fragments downstream and upstream of nahA . pPB41 49 backbone was amplified by oJW2775/oJW2821. Guide RNA template was made by annealing oJW3501/oJW3502.…”

Cryptic protein interactions regulate DNA replication initiation

“…The dnaD23 strain was created using CRISPR/Cas9 gene editing to introduce an A166T mutation in dnaD using a method we developed previously (Burby and Simmons, ; ). A detailed description of the process can be viewed in the supporting information section.…”

Class 2 CRISPR/Cas: an expanding biotechnology toolbox for and beyond genome editing

“…As a genome editing tool (Fig. 3 a), CRISPR/Cas9 system has been successfully applied in virus [ 19 – 21 ], bacteria [ 22 – 25 ], yeast [ 26 – 32 ], plant [ 33 – 37 ], nematode [ 38 ], Drosophila [ 39 ], zebrafish [ 40 ], frog [ 41 ], chicken [ 42 ], mice [ 43 ], sheep [ 44 ], monkey [ 45 ] and human [ 46 ], etc.…”