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.

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.
<ol>
	<li>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&#160;Streptococcus pyogenes Cas9 and protospacer adjacent motif NGG after selecting the &#34;Fasta Target&#34;. Define the parameters to &#34;CRISPR/Cas9&#34; and PAM (protospacer adjacent motif) NGG.</li>
	<li>Based on the results obtained, select protospacers with the best score possible (green arrow), that are located as close as possible to the 5&#180;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.</li>
	<li>Use the lipL32 promoter to express the single guide RNA that contains a variable 20 nucleotide sequence at the 5&#39; end and a conserved dCas9 scaffold sequence. Merge the 20-nt sequence, termed protospacer, to the lipL32 promoter (at its 5&#180; end) and sgRNA scaffold (3&#39;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.9). Check the Supplementary File for the final sgRNA cassette.</li>
	<li>Generate the sgRNA cassette by sequential PCR5 or have it synthesized by a commercial provider.</li>
	<li>After obtaining the cassette, ligate it into pMaOri.dCas9 plasmid at the XmaI restriction site at both ends (cccggg)4.
	<ol>
		<li>Digest both the sgRNA cassette and pMaOri.dCas9 plasmid with XmaI restriction enzyme and proceed to ligation (Figure 1B).</li>
		<li>Perform the cloning steps in the dT auxotrophic E. coli strain &#960;110, due to the pMaOri11 (and by extension, pMaOri.dCas9) origin of replication, R6K-gamma. 
		&#8203;NOTE: For a detailed protocol for ligation and clone selection, refer to previous publications by Fernandes and Nascimento12. 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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Grow pathogenic Leptospira cells at 29 or 37 &#176;C in HAN media13 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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Transform the conjugative E. coli strain &#946;2163, 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.
	<ol>
		<li>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 &#176;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 &#176;C for 1 h and proceed to the plating.</li>
		<li>For electroporation, use electrocompetent cells mixed with 100 ng of plasmid DNA. Use the following parameters for pulse: 1.8 kV, 100 &#937;, and 25 &#181;F. Recover the cells as explained above.</li>
		<li>Plate the transformed donor E. coli cells in LB agar medium supplemented with Diaminopimelic acid (DAP) (0.3 mM) and spectinomycin (40 &#956;g/mL) to select for plasmids.</li>
	</ol>
	</li>
	<li>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).
	<ol>
		<li>Select one colony of E. coli &#946;2163 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 &#176;C.</li>
		<li>The next day, dilute the saturated cultures 1:100 in 10 mL of fresh LB plus DAP (do not include the antibiotic here) until OD420nm of 0.2-0.4. Normally, it takes 2-3 h for E. coli to reach these densities.</li>
	</ol>
	</li>
	<li>Inside a BSL2 biosafety hood, assemble a filtration apparatus by placing a 25 mm diameter, 0.1 &#181;m 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.</li>
	<li>Add 5 mL of Leptospira culture to the funnel. Add a volume of E. coli to constitute the 1:1 proportion based on the OD420nm 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.</li>
	<li>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 &#176;C for 24 h. 
	NOTE: If HAN or supplemented in-house EMJH14 plates are used, E. coli can proliferate and overcome the intended 1:1 proportion, which in turn can decrease conjugation efficiency5.</li>
	<li>After 24 h, recover the filters from the plates and place each individual filter in a 50 mL conical tube.</li>
	<li>Use 1 mL of liquid HAN medium to release the cells from the filter surface by extensive pipetting and vortexing.</li>
	<li>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.</li>
	<li>Spread 100-200 &#956;L of this culture onto HAN plates containing 0.4% inactivated rabbit serum and 40 &#956;g/mL spectinomycin. Incubate plates at 37 &#176;C in a 3% CO2 atmosphere. 
	&#8203;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.</li>
	<li>As a control, dilute cultures at 104 leptospires/mL and add 100 &#956;L onto plates without antibiotic, for monitoring leptospiral growth.</li>
</ol>
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.
<ol>
	<li>Add 100 &#956;L of liquid HAN media to each 1.5 mL microtube to recover mutants. Take at least 3 colonies from each plate.
	<ol>
		<li>With the aid of a micropipette tip, &#34;dig&#34; the agar to retrieve the colonies from the plates since leptospiral colonies can be subsurface. 
		NOTE: Agar is expected to be taken along at this stage. Colonies should be taken from control plates containing empty pMaOri.dCas9 plasmid, and plates with leptospires containing plasmids expressing both dCas9 and single guide RNA, designed for the target gene.</li>
		<li>Dispense the collected colony in 100 &#956;L of HAN media in a 1.5 mL microtube and vigorously homogenize. At this stage, ensure maximal break of the agar integrity to release cells. Vortex the suspension for 10 s.</li>
	</ol>
	</li>
	<li>Visualize the recovered cells by dark field microscopy at a 200-400x magnification by adding a 5 &#181;L drop onto a glass slide and cover the samples immediately with a coverslip.
	<ol>
		<li>Confirm the presence of live and viable leptospires recovered from the colonies.</li>
		<li>After visualization and confirmation of viable leptospires, transfer 100 &#956;L of cells to liquid HAN media containing 40 &#956;g/mL spectinomycin.</li>
	</ol>
	</li>
	<li>After growth in liquid HAN media, evaluate the cultures for the presence of the plasmid with primer pMaOri2 F (ACGCAATGTATCGATACCGAC) and R (ATAGGTGAAGTAGGCCCACCC), which recognize the region that flanks the sgRNA cassette.
	<ol>
		<li>Collect 200 &#956;L of culture, centrifuge (4,000 x g, 15 min), discard the supernatant, and resuspend the resulting pellet in 20 &#956;L of water.</li>
		<li>Use this suspension as a template for additional PCR, without the need for DNA extraction12. 
		NOTE: Cells with pMaOri.dCas9 will render an amplicon of 281 bp, compared to those containing the plasmids with the sgRNA cassette which will render an amplicon of 723 bp.</li>
	</ol>
	</li>
	<li>For confirmation of gene silencing, perform an immunoblot utilizing cell extracts from transconjugants containing only pMaOri.dCas9 (negative control) and pMaOri.dCas9sgRNA.
	<ol>
		<li>Apply the equivalent of 5 x 107 cells per lane of the sodium dodecyl sulfate (SDS) polyacrylamide gel.</li>
		<li>Electrotransfer proteins to a membrane for incubation with appropriate antibodies. Besides the antibody against the target gene for silencing, use another one for loading control.</li>
	</ol>
	</li>
	<li>Keep the mutant cultures in HAN media plus spectinomycin for maintaining the plasmid. If no antibiotics are applied to the media, complete gene silencing can be observed for at least three passages5.</li>
</ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-temperature+protein+G+is+an+essential+virulence+factor+of+Leptospira+interrogans.">High-temperature protein G is an essential virulence factor of Leptospira interrogans.</a> Infections and Immunity. 82 (3), 1123-1131 (2014).</li><li>Lambert, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FlaA+proteins+in+Leptospira+interrogans+are+essential+for+motility+and+virulence+but+are+not+required+for+formation+of+the+flagellum+sheath.">FlaA proteins in Leptospira interrogans are essential for motility and virulence but are not required for formation of the flagellum sheath.</a> Infections and Immunity. 80 (6), 2019-2025 (2012).</li><li>Murray, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leptospira+interrogans+requires+heme+oxygenase+for+disease+pathogenesis.">Leptospira interrogans requires heme oxygenase for disease pathogenesis.</a> Microbes and Infections. 11 (2), 311-314 (2009).</li><li>Ristow, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+OmpA-like+protein+Loa22+is+essential+for+leptospiral+virulence.">The OmpA-like protein Loa22 is essential for leptospiral virulence.</a> PLoS Pathogens. 3 (7), 97 (2007).</li><li>Sasaki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leptospiral+flagellar+sheath+protein+FcpA+interacts+with+FlaA2+and+FlaB1+in+Leptospira+biflexa.">Leptospiral flagellar sheath protein FcpA interacts with FlaA2 and FlaB1 in Leptospira biflexa.</a> PLoS One. 13 (4), 0194923 (2018).</li><li>Gaj, T., Gersbach, C. A., Barbas, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZFN,+TALEN,+and+CRISPR/Cas-based+methods+for+genome+engineering.">ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering.</a> Trends in Biotechnology. 31 (7), 397-405 (2013).</li><li>Cress, B. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+generation+of+CRISPR/dCas9-regulated,+orthogonally+repressible+hybrid+T7-lac+promoters+for+modular,+tuneable+control+of+metabolic+pathway+fluxes+in+Escherichia+coli.">Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli.</a> Nucleic Acids Research. 44 (9), 4472-4485 (2016).</li><li>Zhao, C., Shu, X., Sun, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+gene+knockdown+system+based+on+catalytically+inactive+("dead")+Cas9+(dCas9)+in+Staphylococcus+aureus.">Construction of a gene knockdown system based on catalytically inactive ("dead") Cas9 (dCas9) in Staphylococcus aureus.</a> Applied Environmental Microbiology. 83 (12), (2017).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/dCas9-mediated+multiplex+gene+repression+in+Streptomyces.">CRISPR/dCas9-mediated multiplex gene repression in Streptomyces.</a> Biotechnology Journal. , 1800121 (2018).</li></ol>

The authors have nothing to disclose.

After the early sequencing of pathogenic15,16,17,18 and saprophytic19&#160;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&#39;-NGG-3&#39; 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&#39; 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; &#946;-galactosidase silencing in L. biflexa was validated by the use of X-gal and ONPG (ortho-Nitrophenyl-&#946;-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.

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 &#946;2163 and recipient Leptospira cells, transconjugant recovery, and finally, validation of selected mutant colonies.

<table><tbody><tr><td>0.1 &amp;micro;m pore size mixed cellulose esters membrane</td><td>Millipore</td><td>VCWP02500</td><td>Filtration for bacterial conjugation</td></tr><tr><td>2,6-Diaminopimelic acid (DAP)</td><td>Sigma</td><td>D1377</td><td>Growth of auxotrophic &lt;em&gt;E. coli &lt;/em&gt;&amp;beta;2163</td></tr><tr><td>Agar Noble</td><td>BD &amp;amp; Company</td><td>214230</td><td>Used for preparation of solid EMJH and HAN plates</td></tr><tr><td>Bacto Agar</td><td>BD &amp;amp; Company</td><td>214010</td><td>Used for preparation of solid LB plates</td></tr><tr><td>Clarity Western ECL substrate</td><td>Biorad</td><td>170-5060</td><td>Chemiluminescent substrate</td></tr><tr><td>dNTP set</td><td>Thermo Fisher</td><td>10297-018</td><td>dNTPs for PCR reaction</td></tr><tr><td>Glass Microanalysis Filter Holder</td><td>Millipore</td><td>XX1012530</td><td>Filtration for bacterial conjugation</td></tr><tr><td>Imaging System</td><td>Biorad</td><td>ChemiDoc MP</td><td>Chemiluminescence detection</td></tr><tr><td>LB broth, Miller</td><td>BD &amp;amp; Company</td><td>244620</td><td>Lysogenic liquid medium for &lt;em&gt;E. coli&lt;/em&gt; culturing</td></tr><tr><td>&lt;em&gt;Leptospira &lt;/em&gt;Enrichment EMJH</td><td>BD &amp;amp; Company</td><td>279510</td><td>Supplementation of EMJH media</td></tr><tr><td>&lt;em&gt;Leptospira &lt;/em&gt;Medium Base EMJH</td><td>BD &amp;amp; Company</td><td>279410</td><td>EMJH medium for Leptospira</td></tr><tr><td>Mini-PROTEAN TGX Gels 12%</td><td>Biorad</td><td>4568043</td><td>Used for polyacrylamide gel eletrophoresis</td></tr><tr><td>Optical density reader</td><td>Molecular Devices</td><td>SpectraMax M2</td><td>For optical density measurements of bacterial cultures</td></tr><tr><td>Phosphate Buffered Saline 7.4</td><td>Sigma</td><td>806552</td><td>Saline solution for washing bacterial pellets</td></tr><tr><td>Spectinomycin</td><td>Sigma</td><td>S0692</td><td>Selection of pMaOri backbone plasmids</td></tr><tr><td>Taq DNA Polymerase</td><td>Thermo Fisher</td><td>EP0402</td><td>Enyme, buffer and MgCl2 for PCR reaction</td></tr><tr><td>Thermocycler</td><td>Applied Biosystem</td><td>GeneAmp PCR System 9700</td><td>Used for PCR reaction cycling</td></tr><tr><td>Thymidine (dT)</td><td>Sigma</td><td>T9250</td><td>Growth of auxotrophic &lt;em&gt;E. coli&lt;/em&gt; &amp;pi;1</td></tr><tr><td>XmaI restriction enzyme</td><td>New Englan BioLabs</td><td>R0180L</td><td>Digestion of plasmids and inserts</td></tr></tbody></table>

application of crispr interference (crispri) for gene silencing in pathogenic species of leptospira

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 &#946;2163, 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.

Even though the CG content in Leptospira spp. genomes is typically around 35%; virtually every gene is likely to contain the PAM 5&#39;NGG 3&#39;; 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 &#956;L 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62631/62631fig01.jpg" /> 
Figure 1: Development of dCas9 and sgRNA expressing plasmid. (A) A 20-nt long protospacer, followed by the S. pyogenes dCas9 PAM 5&#39;-NGG-3&#39;, 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62631/62631fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62631/62631fig02.jpg" /> 
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 &#946;2163 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 &#956;m 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 &#176;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 &#956;L of each suspension are seeded onto HAN agar plates containing 0.4% rabbit serum and incubated at 37 &#176;C in 3% CO2. At this stage, DAP is omitted, and as a result, auxotrophic E. coli will not grow. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62631/62631fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62631/62631fig03.jpg" /> 
Figure 3: Representative results for mutants&#39; 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62631/62631fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62631/Supplementary File.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Application of CRISPR Interference CRISPRi for Gene Silencing in Pathogenic Species of Leptospira at JoVE.com

Application of CRISPR Interference CRISPRi for Gene Silencing in Pathogenic Species of Leptospira

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.

Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira

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 ...

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5.0K Views.  United States Department of Agriculture. Mijn naam is Jarlath Nally, en ik maak deel uit van het onderzoeksteam hier in het National Animal Disease Center in Ames, Iowa, en we werken aan leptospirose. Leptospirose is een verwaarloosde ziekte. Het is een wereldwijde ziekte.En echt, we hebben een zeer beperkt begrip van hoe deze unieke groep bacteriën infectie veroorzaken. De presentatie van vandaag gaat dus over het bieden van een overzicht van een nieuw hulpmiddel dat beschikbaar is om de evaluatie van pathogene mechanismen ...

Video: Application of CRISPR Interference CRISPRi for Gene Silencing in Pathogenic Species of Leptospira

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene.

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to visceral pathology. Deciphering the nature of the vector/parasite interactions is of primary importance for better understanding of Leishmania transmission to their hosts. Among the parameters controlling the sand fly vector competence (i.e. their ability to carry and transmit pathogens), parameters intrinsic to these insects were shown to play a key role. Insect immune response, for example, impacts sand fly vector competence to Leishmania. The study of such parameters has been limited by the lack of methods of gene expression modification adapted for use in these non-model organisms. Gene downregulation by small interfering RNA (siRNA) is possible, but in addition to being technically challenging, the silencing leads to only a partial loss of function, which cannot be transmitted from generation to generation. Targeted mutagenesis by CRISPR/Cas9 technology was recently adapted to the Phlebotomus papatasi sand fly. This technique leads to the generation of transmissible mutations in a specifically chosen locus, allowing to study the genes of interest. The CRISPR/Cas9 system relies on the induction of targeted double-strand DNA breaks, later repaired by either Non-Homologous End Joining (NHEJ) or by Homology Driven Repair (HDR). NHEJ consists of a simple closure of the break and frequently leads to small insertion/deletion events. In contrast, HDR uses the presence of a donor DNA molecule sharing homology with the target DNA as a template for repair. Here, we present a sand fly embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 using NHEJ, which is the only genome modification technique adapted to sand fly vectors to date.

Sand Fly Phlebotomus papatasi Embryo Microinjection for CRISPR/Cas9 Mutagenesis

This protocol details the steps of CRISPR/Cas9 targeted mutagenesis in sand flies: embryo collection, injection, insect rearing, and identification as well as selection of mutations of interest.

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to ...

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

Neuroscience

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

Immunology and Infection

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

Biology

Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation

The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including their interplay with significant human pathogens such as the human immunodeficiency virus (HIV). Compared to other immortalized cell lines of myeloid origin, THP-1 cells retain many intact inflammatory signaling pathways and display phenotypic characteristics that more closely resemble those of primary monocytes, including the ability to differentiate into macrophages when treated with phorbol-12-myristate 13-acetate (PMA). The use of CRISPR-Cas9 technology to engineer THP-1 cells through targeted gene knockout (KO) provides a powerful approach to better characterize immune-related mechanisms, including virus-host interactions. This article describes a protocol for efficient CRISPR-Cas9-based engineering using electroporation to deliver pre-assembled Cas9:sgRNA ribonucleoproteins into the cell nucleus. Using multiple sgRNAs targeting the same locus at slightly different positions results in the deletion of large DNA fragments, thereby increasing editing efficiency, as assessed by the T7 endonuclease I assay. CRISPR-Cas9-mediated editing at the genetic level was validated by Sanger sequencing followed by Inference of CRISPR Edits (ICE) analysis. Protein depletion was confirmed by immunoblotting coupled with a functional assay. Using this protocol, up to 100% indels in the targeted locus and a decrease of over 95% in protein expression were achieved. The high editing efficiency makes it convenient to isolate single-cell clones by limiting dilution.

The THP-1 cell line is widely used as a model to investigate the functions of human monocytes/macrophages across various biology-related research areas. This article describes a protocol for efficient CRISPR-Cas9-based engineering and single-cell clone isolation, enabling the production of robust and reproducible phenotypic data.

The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.

Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira

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