Mycobacteriophages infect and kill mycobacteria, several of which M. tuberculosis (Mtb), for example, cause tuberculosis. Although many such phages have been isolated and their genomes sequenced, we have very little insight into how they express their genes in a controlled manner. Earlier, we had reported that the multiple repressor bindings sites present in the genome of phage D29, known as stoperators, can potentially function as promoters. In this study, we have investigated how the mycobacteriophage D29 uses the stoperators positively to control gene expression using a novel CRISPR-cas9 based approach. We targeted six potential stoperators and found that the phage production decreased significantly in the case of five. RT-PCR analysis revealed that CRISPRi targeting of stoperators led to a significant decrease in the expression of genes located immediately downstream of the targeted sites but not upstream. We also obtained evidence to show that these sites function as promoters by performing primer extension assays. We could identify transcription start sites downstream of the stoperators we examined, confirming their ability to act as promoter elements. Therefore, transcription from the D29 genome is initiated from multiple points that appear to coincide with stoperators.