Here, the application of CRISPR interference (CRISPRi) for specific gene silencing in Leptospira species is described. Leptospira cells are transformed by conjugation with plasmids expressing dCas9 (catalytically “dead” Cas9) and a single-guide RNA (sgRNA), responsible for base pairing to the desired genomic target. Methods to validate gene silencing are presented.
Leptospirosis is a global neglected zoonosis, responsible for at least 1 million cases per year and almost 60 thousand deaths. The disease is caused by pathogenic and virulent bacteria of the genus Leptospira, either by direct contact with the bacteria or indirectly by exposure to contaminated water or soil. Domestic and wild animals act as reservoir hosts of infection, shedding leptospires from colonized renal tubules of the kidney, via urine, into the environment. The generation of mutant strains of Leptospira is critical to evaluate and understand pathogenic mechanisms of infection. CRISPR interference (CRISPRi) has proven to be a straightforward, affordable, and specific tool for gene silencing in pathogenic Leptospira. Therefore, the methodological details of obtaining the plasmid constructs containing both dCas9 and guide RNA, delivery of plasmids to Leptospira by conjugation with the E. coli strain β2163, and transconjugant recovery and evaluation, will be described. In addition, the recently described Hornsby-Alt-Nally (HAN) media allows for the relatively rapid isolation and selection of mutant colonies on agar plates.
Leptospirosis is a neglected worldwide zoonosis caused by pathogenic and virulent species of the genus Leptospira. In humans, the disease accounts for more than one million cases and 60,000 deaths per year worldwide1,2. So far, there is no long-term and effective vaccine for the disease. The identification of virulence factors and pathogenic mechanisms is pivotal to the development of better therapeutic and prophylactic strategies. Therefore, the ability to generate genetic mutations and assess the resulting phenotype is critical to functional genomic analysis3.
The construction of mutants in pathogenic Leptospira was considered, until now, inherently inefficient, laborious, costly, and difficult to implement. This scenario drastically changed with the application of the recent CRISPR interference (CRISPRi) to saprophytic4 and pathogenic5 leptospires.
Gene silencing is achieved by the expression of two components: a variant of the CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR associated) enzyme Cas9 from Streptococcus pyogenes, called catalytically dead Cas9 (dCas9) and a single-guide RNA (sgRNA), that can be edited according to the desired target6,7,8. dCas9 protein, when bound to the sgRNA, is directed to specific DNA targets by Watson and Crick base pairing, causing a steric blockage to RNA polymerase elongation, resulting in gene silencing due to the obstructed gene transcription7 (Figure 1).
This manuscript aims to describe the construction of the plasmid for expressing both dCas9 and sgRNA, conjugation between donor E. coli β2163 and recipient Leptospira cells, transconjugant recovery, and finally, validation of selected mutant colonies.
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.
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.13 and Fernandes et al.5.
3. Colony selection and transconjugant growth and validation
NOTE: Colonies should be apparent by day 10. However, they are not too easy to visualize. Normally at this time point, HAN plates are a bit opaque because of the dried cells that were spread and Leptospira colonies can appear as a transparent halo against the whitish background. It is recommended to view the plates at different angles to achieve different light incidence, therefore, making the colonies more apparent. At longer incubation times, colonies can acquire a denser appearance, and in this case, they present as milky halos against a dark background.
Even though the CG content in Leptospira spp. genomes is typically around 35%; virtually every gene is likely to contain the PAM 5'NGG 3'; this motif needs to be considered in the template strand. After inputting the coding sequence of a gene (from start to stop codons), based on the CHOPCHOP results, protospacers must be selected at the minus (-, template) strand. It is important to not include the NGG motif in the 20-nt sgRNA protospacer.
If conjugation is performed with a 1:1 donor:recipient cell proportion, for 24 h on the surface of EMJH agar plates plus DAP, and 200 μL of the recovered bacterial suspension are spread onto HAN plus spectinomycin agar plates, transconjugants colonies should be visible at approximately 8-10 days. Spreading of this volume normally results in 20-40 colonies per plate (Figure 3A). In order to check for cell viability after conjugation, cells can be spread onto HAN plates with no antibiotic selection. In this case, colonies can be observed as soon as 7 days. HAN plates turn pale yellow in a 3% CO2 atmosphere.
After colony picking and growth in liquid media plus spectinomycin, PCR using whole cells and pMaOri2 primers can be used for an initial quality check of the transconjugants (Figure 3B). Leptospiral cells containing the control pMaOri.dCas9 plasmid should result in an amplicon of 281 bp, while those cells containing the plasmid for silencing, that is, containing both dCas9 and sgRNA, should result in a 723 bp amplicon. pMaOri2 F and R primers were designed to flank the XmaI restriction site, which is the site used during sgRNA cassette ligation.
With the confirmation of plasmid presence, cells can be harvested from the media, washed twice with PBS, and then used to prepare a whole-cell extract for immunoblotting. If the silencing occurred, the target proteins, in this case, either LipL32 or both LigA and LigB, should be observed only in the wild type cells and in those containing pMaOri.dCas9; even with higher exposure times, no corresponding proteins should be visible in the cells containing pMaOri.dCas9sgRNA (Figure 3C).
If experiments to assess leptospiral virulence after gene silencing are planned, cultures used for conjugation should be low passage virulent Leptospira. After gene silencing is confirmed, several aliquots can be frozen as a backup. If the silenced gene has a measurable phenotype, e.g., based on previous work with recombinant proteins, cultures can be used for validation and, in this case, cells containing pMaOri.dCas9 only, can be included as a negative control.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 3: Representative results for mutants' evaluation. (A) Colonies from plates containing Leptospira transformed with empty pMaOri.dCas9 (negative control for further experiments) and plasmids pMaOri.dCas9sgRNA (with targeted gene silenced) are picked, vigorously homogenized in liquid HAN and grown in liquid HAN containing spectinomycin. Recombinant cells can be validated by PCR with primers flanking the XmaI site within pMaOri.dCas9. (B) In this case, cells containing pMaOri.dCas9 only resulted in an amplicon of 281 bp, while those cells containing the plasmid for silencing, containing both dCas9 and sgRNA, showed a 723 bp amplicon. After confirmation of the presence of the plasmids, gene silencing was validated by immunoblot analysis. (C) Incubation with antibodies to both target protein and a loading control protein is recommended; in the representative immunoblot, whole-cell extracts from transconjugants containing pMaOri.dCas9 alone or with sgRNA cassettes targeting lipL32 (pMaOri.dCas9sgRNAlipL32) and both LigA and LigB (pMaOri.dCas9sgRNAligAB) genes are displayed. Co-incubation with anti-LipL32, anti-LigAB and anti-LipL41 (non-target, loading control) confirms that the expression of LipL32 protein is abolished in cells containing pMaOri.dCas9sgRNAlipL32 and both LigA and LigB in cells containing pMaOri.dCas9sgRNAligAB. Please click here to view a larger version of this figure.
Supplementary File: 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. Please click here to download this File.
After the early sequencing of pathogenic15,16,17,18 and saprophytic19 Leptospira species, data mining of the genome shed light on several aspects of leptospiral pathogenesis. In most cases, protein function was explored by using the recombinant counterpart of putative leptospiral surface-exposed proteins and subsequent speculation of the native protein function20,21,22,23,24,25,26.
The generation of mutants, and evaluation of their respective phenotype, are key components of functional genomic analysis. Initial attempts to generate mutants in Leptospira spp. were achieved by random transposon mutagenesis27,28,29,30; however, after extensive and laborious analysis for inferring the identity of disrupted genes, it was noted that only 15% of all genes in L. interrogans serovar Manilae were disrupted27. Targeted gene knockout was further achieved by homologous recombination utilizing suicide plasmids to deliver an antibiotic resistance cassette flanked by homologous arms within the desired target31,32.
By applying these technologies, several aspects of leptospiral basic biology and virulence were explored31,33,34,35,36,37. The development of the E. coli–Leptospira conjugative shuttle vector, pMaOri 11, allowed the delivery of components for episomal gene silencing.
It was previously shown that the Cas9-induced double-strand break is lethal to Leptospira spp. and, as an alternative, the catalytically inactive variant of the enzyme, dCas9, can be used to achieve gene silencing in both saprophytic and pathogenic species4, 5. By using the plasmid pMaOri.dCas9 as a backbone for sgRNA cassette ligation, specific and stable gene silencing can be obtained due to the expression of both dCas9 and sgRNA; dCas9-bound sgRNA will lead the protein to the desired target by Watson-Crick base pairing.
For complete gene silencing, the protospacer should be designed based on the template strand of the desired gene so that base pairing of the sgRNA occurs with the coding strand. Based on an average C+G content of 35% in Leptospira spp., the PAM 5'-NGG-3' will occur at least 3 times every 100 bp. Therefore, virtually any gene within the genome of Leptospira will contain at least one PAM. However, if the motif NGG is not found, the alternative NAG motif can be evaluated.
Previous gene silencing techniques, such as zinc fingers and TALE (transcription activator-like effectors), relied on the construction of one different protein to each target, making these techniques laborious and costly38. In the case of CRISPRi, the variable component is the sgRNA, making it necessary to only change the 20 bp at the 5' end. Complete, stable, and targeted gene silencing has been observed not only in Leptospira spp.4,5, but also in other bacteria8,39,40,41.
The development of HAN media13 favored the recovery of mutants by drastically reducing the incubation time for colony formation and allowing Leptospira to grow at 37 oC. However, during the conjugation step, its use is not recommended since E. coli can vigorously proliferate in this media and overcome the intended 1:1 proportion between donor and recipient cells. At this stage, EMJH plus DAP is the better choice, since E. coli replicate poorly in this media. It is worth mentioning that some laboratories make in-house supplemented EMJH, which can contain additional components that might also support the growth of E. coli cells.
The conjugation protocol presented here was optimized for L. interrogans serovar Copenhageni strain Fiocruz L1-130, and it was also proven to be effective in the transformation of a recently isolated pathogenic strain from soil samples5. Initial attempts with different serovars of L. borgpetersenii species indicate lower conjugation efficiencies with the described protocol. Thus, when working with different species/serovars of Leptospira, optimal conditions for conjugation should be determined empirically, considering donor:recipient cell proportions, initial cell densities, conjugation media and time (24 and 48 h). It is reasonable to assume that different Leptospira species and serovars will behave differently with different conjugation protocols.
Even though saprophytic Leptospira colonies are relatively easy to visualize on plates, pathogenic colonies can be more difficult to observe. Normally, by using HAN media supplemented with 0.4% rabbit serum and spectinomycin, transconjugant colonies can be observed at day 10. In our experience, colonies initially present as a transparent halo at the media surface. In the video protocol, denser colonies, after 14 days of growth, are shown since the transparent ones were difficult to film. At this stage, rotating the plate to achieve different light incidence and shifting between white and dark backgrounds can help identify colonies.
For mutant validation, immunoblotting offers a straightforward approach; however, since antibodies are not always available against target proteins, alternative strategies to validate gene silencing can be pursued. Quantitative reverse-transcriptase PCR (qRT-PCR) using primers for the target gene and a constitutive control is effective to validate gene silencing since sgRNA-guided dCas9 is responsible for blockage of gene transcription. If the target gene encodes a clearly defined protein band in protein gels, SDS-PAGE can demonstrate silencing, and as per the lipL32 gene silencing5. If LPS biosynthesis genes are silenced, LPS staining can be employed; in the case of silencing genes encoding for enzymes with well-defined substrates, kinetic assays with chromogenic substrates are valid strategies; β-galactosidase silencing in L. biflexa was validated by the use of X-gal and ONPG (ortho-Nitrophenyl-β-galactoside) substrates4.
After confirmation of gene silencing, experiments can be designed to further evaluate phenotype. Binding assays can be performed in the case of silencing bacterial adhesins; serum-challenge assays confirmed the role of LigA and LigB in serum survival displayed by pathogenic Leptospira5. Mutants can also be used to inoculate animals to assess attenuation of virulence; in this case, animals inoculated with the mutant should be compared to those infected with cells containing pMaOri.dCas9 only.
In conclusion, the current protocol describes the application of CRISPRi for gene silencing in pathogenic Leptospira species using HAN media to facilitate mutant recovery within 10 days. Gene silencing combined with functional genomic analysis will improve our understanding of pathogenic mechanisms of Leptospira, and ultimately leading to the development of better prophylactic strategies for disease control.
The authors have nothing to disclose.
USDA is an equal opportunity provider and employer. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information, and does not imply recommendation or endorsement by the U.S. Department of Agriculture. The Brazilian agency FAPESP (grant 2014/50981-0) financially supported this work; LGVF is funded with a fellowship from FAPESP (2017/06731-8 and 2019/20302-8). The funders had no role in study design, data collection and analysis, decision to publish, or manuscript preparation. The authors also thank Hannah Hill and Alexander Grimes from the USDA Visual Services for filming and editing the video protocol.
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 |
Clarity Western ECL substrate | Biorad | 170-5060 | Chemiluminescent substrate |
dNTP set | Thermo Fisher | 10297-018 | dNTPs for PCR reaction |
Glass Microanalysis Filter Holder | Millipore | XX1012530 | Filtration for bacterial conjugation |
Imaging System | Biorad | ChemiDoc MP | Chemiluminescence detection |
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 |
Mini-PROTEAN TGX Gels 12% | Biorad | 4568043 | Used for polyacrylamide gel eletrophoresis |
Optical density reader | Molecular Devices | SpectraMax M2 | For optical density measurements of bacterial cultures |
Phosphate Buffered Saline 7.4 | Sigma | 806552 | Saline solution for washing bacterial pellets |
Spectinomycin | Sigma | S0692 | Selection of pMaOri backbone plasmids |
Taq DNA Polymerase | Thermo Fisher | EP0402 | Enyme, buffer and MgCl2 for PCR reaction |
Thermocycler | Applied Biosystem | GeneAmp PCR System 9700 | Used for PCR reaction cycling |
Thymidine (dT) | Sigma | T9250 | Growth of auxotrophic E. coli π1 |
XmaI restriction enzyme | New Englan BioLabs | R0180L | Digestion of plasmids and inserts |