Journal: Frontiers in Immunology

Article Title: CRISPR/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate

doi: 10.3389/fimmu.2022.867195

Figure Lengend Snippet: Schematic representation of the pKLM-CRISPR vector. The pKLM-CRISPR vector, contains the expression cassette for codon optimized cas9 gene and a cassette for expression of sgRNA, both under regulation of pUV15tetO, a tetracycline-inducible promoter. The repressor tetR is expressed constitutively by the pimyc promoter. Transcriptional terminators (T) were introduced after each expression cassette. The restriction sites used for cloning are also indicated. The two BbsI sites were introduced to facilitate the cloning of the crRNA sequences upstream of tracrRNA. The vectors also contain a kanamycin resistance marker (KanR); origin of replication in E. coli (oriC) and mycobacteria (oriM).

Article Snippet: Codon-optimized cas9 expression cassette, the Tet regulator ( tetR ) cassette and the tracrRNA sequences (Genscript, Piscataway, NJ, USA) were sequentially cloned into pJH152 (a kind gift from Dr. Stewart Cole, EPFL, Lausanne, France) using the restriction enzymes NotI/ClaI, HpaI and NheI (New England Biolabs, Ipswich, MA, USA), respectively, originating pKLM-CRISPR ( ).

Techniques: CRISPR, Plasmid Preparation, Expressing, Cloning, Marker