CRISPR Interference-Based Gene Silencing: A Technique for Targeted Repression of Gene Function in Pathogenic Leptospira
Transcript
CRISPR interference or CRISPRi, uses a deactivated variant of Cas9, dCas9, for targeted gene silencing, which represses gene function.
To perform CRISPRi, use a shuttle vector construct – a plasmid containing origins of replication corresponding to both E. coli and Leptospira bacteria, facilitating propagation in both species.
The vector also contains a sequence specific for the dCas9 gene and a single-guide RNA or sgRNA, to recognize the target sequence.
Initially, the shuttle vector is present inside donor E. coli. Supplement the donor E. coli culture with the host Leptospira to facilitate conjugation. Conjugation establishes direct cell-to-cell contact between organisms, facilitating shuttle plasmid uptake by Leptospira. Inside the cell, the plasmid construct produces sgRNA sequences and expresses dCas9 proteins.
The sgRNA contains a protospacer region that recognizes its complementary target region on the Leptospira DNA, near to the protospacer adjacent motif or PAM – a short recognition sequence on the host DNA. This helps dCas9 form a complex with the sgRNA at the target site downstream to the gene transcription start site.
dCas9 from the sgRNA-dCas9 complex does not have the catalytic activity to cleave DNA strands, instead, it acts as a physical barrier for mRNA-forming RNA polymerase enzyme movement, eventually inhibiting gene transcription. Use the gene-silenced Leptospira for further testing.
