This article discusses the use of CRISPR interference (CRISPRi) for targeted gene silencing in Leptospira bacteria. It details the methodology for constructing a shuttle vector and the process of conjugation between E. coli and Leptospira to facilitate gene repression.
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.
For conjugation, Leptospira cells are grown at 29 or 37 degrees Celsius in HAN media. One day before conjugation, donor E. coli cells are grown in LB media supplemented with Diaminopimelic acid - DAP, and spectinomycin.
Next, in the day of conjugation, grow the saturated E. coli cultures in LB plus DAP. Inside a biosafety hood, assemble the filtration apparatus by placing the membrane filter on top of the glass base, followed by 15-mL glass funnel. Both pieces are held together by the spring clamp. Then, connect the glass to a vacuum pump. Add 5 ml of Leptospira cultures to the funnel.
Next, add the E. coli β2163 strain containing the plasmid of interest. The volume will vary according to the optical density of the culture. We are aiming to 1:1 cell proportion. Turn the vacuum pump. Now, liquids will get through the membrane, and cells will be concentrated on top of it.
Take the filter, and place it onto EMJH plate plus DAP, bacterial side up. Incubate the plates at 29 degrees Celsius for 24 hours. Then, recover the filters from the plates, and place it into 50-mL conical tube.
Use 1 mL of liquid HAN media to recover the cells from the filter by pipetting. Visualize the recovered cells by dark-field microscopy.
At this stage, equivalent numbers of E. coli and Leptospires can be seen. 100 to 200 microliters are used to spread the bacteria on two HAN plates containing spectinomycin. At this stage, DAP is omitted, and E. coli will not grow. Plates are incubated at 37 degrees Celsius and 3% CO2. Colonies should be apparent between 8 to 10 days.
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