CRISPR Interference-Based Gene Silencing: A Technique for Targeted Repression of Gene Function in Pathogenic Leptospira

Overview

This video demonstrates a CRISPR interference-based gene silencing in pathogenic Leptospira. Targeted repression of gene expression using a CRISPR/Cas system that blocks mRNA formation helps identify candidate genes for pathological and pharmacological studies.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Protospacer definition and plasmid construction

NOTE: In this section, the first step of selecting appropriate protospacers for constructing the sgRNA and further ligation into pMaOri.dCas9, is described (Figure 1). This protospacer sequence comprises of a 20 nucleotides sequence against the desired target.

1. Obtain the nucleotide sequence of the gene of interest for silencing at GenBank (https://www.ncbi.nlm.nih.gov/genbank). Submit it to the CHOPCHOP webserver (http://chopchop.cbu.uib.no/), with parameters defined for Streptococcus pyogenes Cas9 and protospacer adjacent motif NGG after selecting the "Fasta Target". Define the parameters to "CRISPR/Cas9" and PAM (protospacer adjacent motif) NGG.
2. Based on the results obtained, select protospacers with the best score possible (green arrow), that are located as close as possible to the 5´end of the coding region and, most importantly, are contained in the template (minus) strand since the sgRNA must pair to the coding strand of the gene for complete gene silencing.
   NOTE: The NGG motif is not included in the final sgRNA sequence.
3. Use the lipL32 promoter to express the single guide RNA that contains a variable 20 nucleotide sequence at the 5' end and a conserved dCas9 scaffold sequence. Merge the 20-nt sequence, termed protospacer, to the lipL32 promoter (at its 5´ end) and sgRNA scaffold (3'end) (Figure 1B).
   NOTE: For a well-defined lipL32 promoter, utilize the promoter region comprising -334 to the TSS (Transcription Start Site, based on Zhukova et al.). Check the Table 1 for the final sgRNA cassette.
4. Generate the sgRNA cassette by sequential PCR or have it synthesized by a commercial provider.
5. After obtaining the cassette, ligate it into pMaOri.dCas9 plasmid at the XmaI restriction site at both ends (cccggg).
   1. Digest both the sgRNA cassette and pMaOri.dCas9 plasmid with XmaI restriction enzyme and proceed to ligation (Figure 1B).
   2. Perform the cloning steps in the dT auxotrophic E. coli strain π1, due to the pMaOri (and by extension, pMaOri.dCas9) origin of replication, R6K-gamma.
      ​NOTE: For a detailed protocol for ligation and clone selection, refer to previous publications by Fernandes and Nascimento. sgRNA-guided dCas9 will bind to the coding strand of the selected gene of interest and, therefore, will obstruct RNA polymerase elongation (Figure 1C), resulting in gene silencing.

2. Leptospira transformation by conjugation

NOTE: A graphical scheme of this step is presented in Figure 2. To make HAN media and HAN plates, refer to Hornsby et al. and Fernandes et al.

1. Grow pathogenic Leptospira cells at 29 or 37 °C in HAN media under agitation by diluting a saturated culture in fresh HAN at 1:100; typically, L. interrogans serovar Copenhageni strain Fiocruz L1-130 takes 4-6 days to reach the appropriate cell density.
   1. Ensure cultures reach an O.D. of 0.2-0.4 at 420 nm (2 to 5 x 10⁸ cells/mL) before using for conjugation.
      NOTE: Since HAN media changes color as cell densities increase (due to phenol red contained in DMEM media), centrifuge (4,000 x g, 15 min, room temperature) 1 mL of the culture media to remove leptospires and apply supernatant as a blank for measuring the O.D.
2. Transform the conjugative E. coli strain β2163, auxotrophic for Diaminopimelic acid (DAP), with the plasmid pMaOri.dCas9 containing the sgRNA cassette. For E. coli transformation, use either heat-shock protocols or electroporation. Include transformation with the plasmid pMaOri.dCas9 with no sgRNA cassette as a control.
   1. For heat-shock transformation, mix the plasmid DNA (100 ng) with chemically competent E. coli cells and incubate on ice for 30 min. Perform heat shock at 42 °C for 90 s and place it again on ice for 5 min. Recover the cells by adding 1 mL of LB media, incubate at 37 °C for 1 h and proceed to the plating.
   2. For electroporation, use electrocompetent cells mixed with 100 ng of plasmid DNA. Use the following parameters for pulse: 1.8 kV, 100 Ω, and 25 µF. Recover the cells as explained above.
   3. Plate the transformed donor E. coli cells in LB agar medium supplemented with Diaminopimelic acid (DAP) (0.3 mM) and spectinomycin (40 μg/mL) to select for plasmids.
3. For conjugation, select one colony from each plate one day before the day of conjugation (which is determined by monitoring the O.D. of cultures of leptospires).
   1. Select one colony of E. coli β2163 from the empty pMaOri.dCas9, and one from pMaOri.dCas9sgRNA plates. Allow them to grow overnight in 10 mL of LB plus DAP and spectinomycin at 37 °C.
   2. The next day, dilute the saturated cultures 1:100 in 10 mL of fresh LB plus DAP (do not include the antibiotic here) until OD_420nm of 0.2-0.4. Normally, it takes 2-3 h for E. coli to reach these densities.
4. Inside a BSL2 biosafety hood, assemble a filtration apparatus by placing a 25 mm diameter, 0.1 µm pore size, mixed cellulose esters membrane filter on the top of the glass base. Place a 15 mL glass funnel on the top and hold both pieces with spring clamps. Connect the glass to a vacuum pump and add the cultures to the funnel for filtration.
5. Add 5 mL of Leptospira culture to the funnel. Add a volume of E. coli to constitute the 1:1 proportion based on the OD_420nm values of both cultures. Turn on the vacuum pump and concentrate cells by filtration. After cell concentration in the membrane filter, carefully retrieve it. Ensure medium is filtered through the membrane.
   NOTE: Filtration takes 5 to 10 min.
6. Place the filter on a commercially available EMJH plate (see Table of Materials) supplemented with DAP (0.3 mM). Ensure that bacteria side is up. Incubate the plates at 29 °C for 24 h.
   NOTE: If HAN or supplemented in-house EMJH plates are used, E. coli can proliferate and overcome the intended 1:1 proportion, which in turn can decrease conjugation efficiency.
7. After 24 h, recover the filters from the plates and place each individual filter in a 50 mL conical tube.
8. Use 1 mL of liquid HAN medium to release the cells from the filter surface by extensive pipetting and vortexing.
9. Visualize the recovered mixed bacterial solutions by dark field microscopy to check for cell viability and motility, and Leptospira:E. coli proportions.
   NOTE: At this stage, equivalent numbers of E. coli and Leptospira can be seen.
10. Spread 100-200 μL of this culture onto HAN plates containing 0.4% inactivated rabbit serum and 40 μg/mL spectinomycin. Incubate plates at 37 °C in a 3% CO₂ atmosphere.
    strong>NOTE: Normally, L. interrogans serovar Copenhageni strain Fiocruz L1-130 cells form colonies in 5-7 days on control plates and in 8-10 days on spectinomycin plates. At this stage, E. coli will not grow since they are auxotrophic for DAP.
11. As a control, dilute cultures at 10⁴ leptospires/mL and add 100 μL onto plates without antibiotic, for monitoring leptospiral growth.

Representative Results

The sgRNA cassette should be: cccgggGAACAAGAAAGAGTCAGAGAATTATTGAAGAGATACTCTTATACTACCGTCTTTGGAAGAATTTTCGCATGGATTTTAGATTTGCTGGACTGGTTGAAGCGATTTTTTCAAAAAAAATAATCAATTTGTGTCTGAGATTTGAAAACGCTTGTTTGATAGTTTTTTAAGAATTTCTGATGTTTCAATCGTATAGAAATTCTAAATTTAGAAATCATCCTTTACTTTTCTCTAAGACTTATATAACAATCGCTTTAAACTCAAATTATAATCTTTCAGATAAAAAATTATTCAATATTGATTTACAAAAAATTCCTAAGTTCATACCGTGATTTTCNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTcccggg Bold nucleotides refer to the lipL32 promoter and underlined nucleotides refer to the dCas9 scaffold. XmaI restriction sites (cccggg) are included at both ends, for ligation at pMaOri.dCas9 plasmid.

Table 1: Single guide RNA (sgRNA) cassette sequence. The sgRNA transcription is directed by the constitutive lipL32 promoter (bold nucleotides). sgRNA is composed of 20 nucleotides referring to the protospacer, responsible for base pairing to the coding strand of the target gene, and dCas9 scaffold sequence (underlined nucleotides). XmaI restriction sites (cccggg) are included at both ends for ligation at pMaOri.dCas9 plasmid.

Figure 1: Development of dCas9 and sgRNA expressing plasmid. (A) A 20-nt long protospacer, followed by the S. pyogenes dCas9 PAM 5'-NGG-3', is selected within the template strand of the target gene so the subsequent sgRNA can perform Watson and Crick base pairing to the corresponding coding strand, resulting in complete gene silencing. (B) The sgRNA cassette is composed of the lipL32 promoter, 20-nt protospacer, and dCas9 scaffold. The pMaOri.dCas9 plasmid is used as a backbone for sgRNA cassette ligation at the XmaI restriction site. The resulting plasmid, termed pMaOri.dCas9sgRNA is delivered to leptospires, and the expression of both dCas9 and sgRNA is responsible for the gene silencing. (C) sgRNA-directed dCas9 acts as a physical barrier to RNA polymerase elongation, therefore, hampering transcription. 

Figure 2: Schematic representation of conjugation protocol. The desired Leptospira species is grown in HAN media, under agitation, until O.D. of 0.2-0.4 (mid-log phase) at 420 nm. One day before conjugation, a colony of recombinant donor E. coli β2163 containing the plasmid of interest is picked from LB+DAP+Spc agar plates, as cells are grown overnight in liquid LB with the same supplementation. The next day, saturated E. coli cultures are diluted in LB plus DAP and grown until O.D. of 0.2-0.4 at 420 nm. Both donor E. coli and recipient Leptospira are mixed at 1:1 cell proportion onto the surface of a 0.1 μm filter by a filtration apparatus under negative pressure. Then, filters are placed on top of the EMJH agar plates supplemented with DAP, and incubation proceeds for 24 h at 29 °C. The use of EMJH limits E. coli proliferation, and the intended 1:1 proportion is maintained. Bacteria are recovered from filters by pipetting with 1 mL HAN media, and suspensions are visualized under darkfield microscopy. Finally, 100-200 μL of each suspension are seeded onto HAN agar plates containing 0.4% rabbit serum and incubated at 37 °C in 3% CO₂. At this stage, DAP is omitted, and as a result, auxotrophic E. coli will not grow.

Materials

Name	Company	Catalog Number	Comments
0.1 µm pore size mixed cellulose esters membrane	Millipore	VCWP02500	Filtration for bacterial conjugation
2,6-Diaminopimelic acid (DAP)	Sigma	D1377	Growth of auxotrophic E. coli β2163
Agar Noble	BD & Company	214230	Used for preparation of solid EMJH and HAN plates
Bacto Agar	BD & Company	214010	Used for preparation of solid LB plates
Glass Microanalysis Filter Holder	Millipore	XX1012530	Filtration for bacterial conjugation
LB broth, Miller	BD & Company	244620	Lysogenic liquid medium for E. coli culturing
Leptospira Enrichment EMJH	BD & Company	279510	Supplementation of EMJH media
Leptospira Medium Base EMJH	BD & Company	279410	EMJH medium for Leptospira
Optical density reader	Molecular Devices	SpectraMax M2	For optical density measurements of bacterial cultures
Spectinomycin	Sigma	S0692	Selection of pMaOri backbone plasmids
Thymidine (dT)	Sigma	T9250	Growth of auxotrophic E. coli π1
XmaI restriction enzyme	New Englan BioLabs	R0180L	Digestion of plasmids and inserts