Bacillus subtilis is a very atractive model microorganism to study bacterial metabolism, gene regulation and bacterial differentiation. Several methods for genetic manipulation of Bacillus subtilis genome have been developed. Therefore, B. subtilis is a promising host for application of metabolic engineering approaches. Successful gene expression and regulation are crucial for industrial applications. Development of gene tools enables transfer of genes to new hosts, deletion of undesirable genes and regulation of gene expression. In 2013, a new genome editing method, CRISPR/Cas9, was developed, which is based on bacterial antiviral immune response. It is an effective, simple and cheap genome-editing method. Limitation for CRISPR/Cas9 method is that it causes a double strand break in DNA, which results in gene inactivation or modification. For purposes of gene inhibition, a modification in Cas9 was made and a novel CRISPRi/dCas9 method was developed. This method enables regulation of gene expression on transcription level. CRISPRi/dCas9 method is useful for studying expression of essential genes. In this work, we used CRISPRi/dCas9 method to downregulate expression of GFP in B. subtilis. We observed decrease in green fluorescence emission, thus lower expression of GFP was confirmed in the culture treated with CRISPRi/dCas9. Therefore, we proved that CRISPRi/dCas9 is a useful method for genetic manipulation of B. subtilis.
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