Targeted Integration of Genetic Elements into The Brucella melitensis 16M?vjbR Genome Using CRISPR-Guided Transposases 9350

Abstract Description The SPIKE (Synthetic Programmable bacteria for Immune-directed Killing in tumor Environments) chassis (B. melitensis 16M?vjbR) has been shown to be a powerful vector for the expression and trafficking of therapeutic molecules for cancer treatment. However, genetic manipulation of the organism has been difficult, and expression of therapeutic molecules has relied on a plasmid-based approach—which has many limitations. Genomic integration of synthetic constructs circumvents these limitations and vastly expands the therapeutic opportunity for this chassis. INTEGRATE, which deploys CRISPR-guided transposases for targeted genomic insertions, has been shown to have high efficacy in many organisms. Therefore, this investigation aimed to evaluate use of the INTEGRATE system in the SPIKE chassis through targeted insertion of the genes mCherry and ampR. Guide RNA sequences were chosen to target key genes of the SPIKE chassis and cloned into two INTEGRATE plasmid variants, which contained either mCherry or ampR for genome insertion. The plasmids were found to function effectively in the microbe, producing desired mutant strains. Genomic integration of mCherry or ampR was confirmed by whole genome sequencing. Phenotypic differences from the parent strain were validated through growth curve analysis. In conclusion, INTEGRATE was successfully utilized in the SPIKE chassis and we expect this finding to have profound implications for the future of bacterial therapeutics. Funding Sources ARPA-H Topic Categories Technological Innovations in Immunology (TECH)