PROCESS FOR CONSTRUCTION OF A SINGLE VECTOR COMPRISING CAS9 AND SGRNA FOR MYCOBACTERIAL GENOME MODIFICATIONS

The present invention relates to a process for constructing a single vector comprising cas9 and sgRNA ("all-in-one" vector) for mycobacterial genome modifications, comprising the steps of (a) providing the expression cassette of cas9 and the tracrRNA expression cassette with codons optimized for expression in mycobacteria and clone into a cloning vector; (b) sequentially cloning the sequences optimized in step (a) into a mycobacterial bridge vector; (c) selecting targeting sequences (crRNAs) specific to the gene of interest; (d) inserting the crRNAs selected in step (c) into the mycobacterial expression vector, upstream of the tracrRNA sequence, at position +1 downstream of an inducible promoter for the construction of the sgRNA cassette; and (e) adding at the end of each expression cassette a transcriptional terminator.