CRISPR Interference for Targeted Gene Silencing in Mycobacteria PDF

**CRISPR Interference (CRISPRi) for Targeted Gene Silencing in Mycobacteria** **Abstract** The genetic basis for Mycobacterium tuberculosis pathogenesis is incompletely understood. One reason for this knowledge gap is the relative difficulty of genetic manipulation of M. tuberculosis. To close thi...

**CRISPR Interference (CRISPRi) for Targeted Gene Silencing in Mycobacteria** **Abstract** The genetic basis for Mycobacterium tuberculosis pathogenesis is incompletely understood. One reason for this knowledge gap is the relative difficulty of genetic manipulation of M. tuberculosis. To close this gap, we recently developed a robust CRISPR interference (CRISPRi) platform for programmable gene silencing in mycobacteria. In this chapter, we: (1) discuss some of the advantages and disadvantages of CRISPRi relative to more traditional genetic approaches; and (2) provide a protocol for the application of CRISPRi to reduce transcription of target genes in mycobacteria. **Introduction** The past 20years have seen dramatic improvements in our ability to genetically manipulate Mycobacterium tuberculosis. These improvements have advanced our understanding of this pathogen. While powerful, each genetic method has advantages and disadvantages. For example, current genetic approaches in M. tuberculosis include promoter replacement and inducible protein degradation systems that allow the regulation of target protein levels over two orders of magnitude \[1--3\], but can take months to target a single gene. Similarly, several methodologies \[4, 5\] now allow for facile gene deletion in M. tuberculosis, but these methods are necessarily restricted to the analysis of genes nonessential for in vitro growth and remain slow to implement. To increase throughput, Kenan Murphy and colleagues developed ORBIT \[6\], a method that dramatically improves the efficiency of M. tuberculosis genetic engineering. While it is a major advance, this method in its present form does not yet scale to parallelized genome-wide genetic manipulation. To work at scale, Transposon-sequencing (TnSeq) allows the simultaneous assessment of hundreds of thousands of loss-of function mutants. But as with gene deletion, TnSeq as currently implemented in M. tuberculosis is limited to analysis of genes non essential for in vitro growth \[7, 8\]. Lastly, none of these methods provide a simple mechanism to simultaneously modulate multiple genes in order to elucidate genetic interactions. To complement existing genetic tools, we recently developed an optimized CRISPR interference (CRISPRi) system for targeted gene silencing in mycobacteria \[9\] (Fig. 1). Unlike most other CRISPRi applications which utilize a Cas9 enzyme derived from Streptococcus pyogenes (SpyCas9) \[10, 11\], we found a Cas9 enzyme derived from S. thermophilus (Sth1Cas9) to have superior performance characteristics (magnitude of target gene knockdown and reduced toxicity) in Mycobacterium smegmatis \[9\]. In this system, the protein dCas9 (with two mutations that disable nuclease activity, thus "dead" or dCas9) is guided to the target gene by a chimeric RNA called a single guide RNA(sgRNA)\[12\]. Targeting specificity is determined both by base pairing of the sgRNA and target DNA, as well as a short DNA motif (protospacer adjacent motif \[PAM\]) within the target DNA sequence. The PAM is a 2--8 base pair sequence located immediately downstream of the sgRNA target sequence \[13--15\]. PAM recognition is an obligate first step for dCas9 binding---recognition of the PAM by dCas9 destabilizes the adjacent DNA duplex, thereby allowing interrogation of the DNA target by the sgRNA \[16, 17\]. Binding of the dCas9--sgRNA complex to the target gene results in transcriptional interference by blocking RNA polymerase promoter access or transcription elongation \[10, 11\]. The M. tuberculosis CRISPRi system \[9\] was further engineered to be inducible by two alternative small molecules (anhydrotetracycline or doxycycline), thereby allowing the facile manipulation of M. tuberculosis genes, be they essential or nonessential for in vitro growth. The efficient cellular and tissue penetration of doxycycline \[18\] should allow CRISPRi-mediated control of the M. tuberculosis transcriptome in numerous experimental settings, including axenic in vitro culture, ex vivo M. tuberculosis infected macrophages, and in vivo animal infection models. An additional advantage of the Sth1 dCas9 CRISPRi system is that the magnitude of target gene silencing is tunable, either by varying targeted PAM "strength" \[9\] or by varying the length of the sgRNA targeting sequence \[10\]. This allows for rheostat-like control of target gene production spanning two orders of magnitude \[9, 10\]. Tunability enables the hypomorphic or partial silencing of target gene production to create an allelic series, thereby enabling the study of interactions (chemical and genetic) between in vitro essential genes \[9, 19\]. Lastly, CRISPRi is scalable. With advances in array-based synthesis, generating large pools of unique sgRNA targeting sequences is fast and inexpensive. Altogether, these unique features set the stage to develop CRISPRi as a powerful new genetic method in M. tuberculosis. In the next sections, we discuss the application and limitations of this approach for targeted gene silencing in M. tuberculosis. **Materials** **Design of CRISPRi sgRNA Targeting Sequence Oligonucleotides (sgRNA Oligos)** 1\. sgRNA oligos: There will be two oligos for each sgRNA to be cloned. Resuspend each oligo in deionized water to a concentration of 100 μM. **Annealing sgRNA Oligos for Ligation into the CRISPRi Plasmid Backbone** 1\. PCR tubes. 2\. Oligos (top and bottom). 3\. Oligo Annealing Buffer: 50 mM Tris pH 7.5, 50 mM NaCl, 1 mMEDTA. **BsmBI-Digestion of the CRISPRi Plasmid Backbone** 1\. BsmBI-v2 (10,000 U/mL; NEB). 2\. NEBuffer 3.1 (supplied with BsmBI-v2). 3\. plJR965 (Add gene \#115163): the CRISPRi backbone for M. tuberculosis. This plasmid contains an Sth1 dCas9 allele, an sgRNA scaffold, a Tet repressor (TetR) codon optimized for expression in M. tuberculosis, an L5 integrase, a kanR gene that confers kanamycin resistance, and an E. coli origin of replication (Fig. 2). dCas9 and the sgRNA are expressed from a TetR regulated promoter that is induced in the presence of anhydro tetracycline (ATc) or doxycycline. The plasmid integrates into the mycobacterial chromosome at the L5 attB site. See notes at the end of each section for modifications when performing CRISPRi in M. smegmatis. 4\. QIA quick Gel Extraction Kit (Qiagen). 5\. TAE buffer. 6\. Molecular biology grade agarose. 7. DNA intercalating dye for visualization (such as SYBR Safe, Thermo Fisher Scientific). **Ligation of the Annealed sgRNA Oligos into the CRISPRi Backbone** 1\. T4 DNA Ligase (2,000,000 U/mL; NEB). 2\. 10 T4DNALigase Buffer. 3\. Chemically competent E. coli for cloning. 4\. LB agar. 5\. Kanamycin. 6\. QIA prep Spin Miniprep Kit (Qiagen) or equivalent. 7\. Oligonucleotide used to Sanger sequence plasmids containing the cloned sgRNA: 5'-TTCCTGTGAAGAGCCATTGATAATG-3'. **Preparation of Electrocompetent Mycobacteria** 1\. M. tuberculosis. 2\. T-25 tissue culture flasks. 3\. 7H9-OADC-Tween80: Dissolve 4.7 g Difco Middlebrook 7H9 Broth powder (BD) in 893.5 mL deionized water and autoclave. Cool to 50 C and then aseptically add 4 mL of filter sterilized 50% (v/v) glycerol, 2.5 mL of filter sterilized 20% (v/v) Tween80, and 100 mL of BBL Middlebrook OADC Enrichment (BD). 4\. 125 mL screw cap Erlenmeyer flask. 5\. Sterile 10% glycerol. **Electroporation of CRISPRi Constructs into Electrocompetent Mycobacteria** 1\. 2mm electroporation cuvettes (Bio-Rad). 2\. 7H10-OADC-Tween80 plates with 20 μg/mL kanamycin: Dissolve 19 g Difco Middlebrook 7H10 Agar powder (BD) in 887.5 mL deionized water and autoclave. Cool to 50 C and then aseptically add 4 mL of filter sterilized 50% (v/v) glycerol, 2.5 mL of filter sterilized 20% (v/v) Tween80, 100 mL of BBL Middlebrook OADC Enrichment (BD), and 400 μL of a 50 mg/mL solution of kanamycin. Mix solution and pour plates. **Quantification of Transcriptional Knockdown by qRT PCR** 1\. T-25 tissue culture flasks. 2\. Anhydrotetracycline (ATc). A 10,000 stock solution can be made by dissolving ATc powder in methanol at a concentration of 1 mg/mL. 3\. Aluminum foil. 4\. GTC Buffer: Dissolve 180 g guanidine thiocyanate, 1.5 g N lauryl sarcosine, and 2.2 g sodium citrate in 297.9 mL water at 37 C.Add 2.1 mLof β-mercaptoethanol. Use within 48 h. 5\. TRIzol Reagent. 6\. Lysing Matrix B, 2 mL tube. 7\. Chloroform. 8\. RNA isolation kits (Zymo Direct-zol RNA Miniprep or equivalent). 9\. cDNA synthesis kits (Invitrogen SuperScript IV First-Strand Synthesis System or equivalent). 10\. Appropriate oligonucleotides for qPCR amplicon generation and quantification. **Design of CRISPRi-Resistant Complementation Constructs** 1\. Gibson assembly oligos. Used to PCR amplify and provide DNA over hangs to clone the complementation gene of interest into an appropriate expression vector. 2\. Oligos to generate CRISPRi-resistance mutations. Designed to include DNA overhangs for Gibson assembly and to introduce synonymous mutations into the gene of interest that abrogate dCas9-sgRNA binding. 3\. Q5 High-Fidelity 2 Master Mix (NEB) or equivalent. Used to amplify DNA fragments for complementation constructs. 4\. NE Builder HiFi DNA Assembly Master Mix (NEB). **Targeting Multiple Genes for Transcriptional Silencing with CRISPRi** 1\. sgRNA oligos targeting genes of interest. 2\. Q5 High-Fidelity 2 Master Mix (NEB) or equivalent. Used to amplify \[promoter-sgRNA-terminator\] cassette from individual CRISPRi plasmids. 3\. 1 MDTT(Millipore Sigma).\ 4. 10 mMATP(NEB). 5\. SapI (10,000 U/μL; NEB). 6\. 10 CutSmart Buffer. 7\. T4 DNA ligase (2,000,000 U/mL; NEB). 8\. Oligos to amplify \[promoter-sgRNA-terminator\] cassette and to add SapI digestion DNA overhangs for cloning into the CRISPRi backbone. 9\. LB agar. 10\. Kanamycin. 11\. Oligos to Sanger sequence Golden Gate assembled plasmids. 12\. GG\_seq\_1: 5'-TGCGGCGCTTTTTTTTTTGAATTC-3'. GG\_seq\_2: 5'-CTGCGTTATCCCCTGATTCTG-3'. **CRISPRi Transcriptional Interference in M. smegmatis** 1\. M. smegmatis mc2155. 2\. plJR962 (Add gene \#115162): the CRISPRi backbone for M. smegmatis (Fig. 2). This plasmid is identical to plJR965 except that it encodes a TetR expressed at higher levels than plJR965 (unpublished data). Elevated TetR expression minimizes leaky dCas9 and sgRNA expression in the absence of ATc but still allows for high-level target gene repression in M. smegmatis. 3\. ADC Enrichment: Dissolve 50 g of Bovine Serum Albumin Fraction V, 20 g dextrose, and 8.5 g sodium chloride in 900 mL of deionized water, gently stirring and heating to 37 C. Once dissolved, remove from heat and add 30 mg of catalase. Stir to dissolve, then supplement the volume to 1 L with deionized water and filter sterilize. 4\. 7H9-ADC-Tween80: Dissolve 4.7 g Difco Middlebrook 7H9 Broth powder (BD) in 893.5 mL deionized water and auto clave. Cool to 50 C and then aseptically add 4 mL of filter sterilized 50% (v/v) glycerol, 2.5 mL of filter sterilized 20% (v/v) Tween80, and 100 mL of ADC Enrichment. 5\. 7H10-ADC-Tween80 plates with 20 μg/mL kanamycin: Dis solve 19 g Difco Middlebrook 7H10 Agar powder (BD) in 887.5 mL deionized water and autoclave. Cool to 50 C and then aseptically add 4 mL of filter sterilized 50% (v/v) glycerol, 2.5 Ml of filter sterilized 20% (v/v) Tween80, 100 mLofADC Enrichment, and 400 μL of a 50 mg/mL solution of kanamycin. Mix solution and pour plates. 6\. 30 mL inkwell (VWR). 7\. 250 mL inkwell (VWR). **Design of CRISPRi sgRNA Targeting Sequence Oligonucleotides (sgRNA Oligos)** Here we describe the sgRNA design work-flow. To assist in this process, we provide a web-based sgRNA design tool at: 1\. CRISPRi shows strong targeting orientation dependency. sgRNAs should be designed to target the non-template strand of the target gene ORF or promoter---this is the strand that is displaced as ssDNA during RNA polymerase elongation during transcription (Fig. 3) \[20\]. Targeting the template strand shows minimal and variable knockdown when targeting the ORF \[9, 10\], although it can be effective when targeting the promoter \[21\]. 2\. PAM choice: the consensus PAM sequence for Sth1 dCas9 is 5'-NNAGAAW-3' \[13\]. Sth1 dCas9 can recognize noncanonical PAMs with mutations within this consensus sequence at the cost of the magnitude of target gene knockdown \[9\]. Importantly, this PAM complexity provides a facile method of tuning target gene knockdown to generate an allelic series. We have ranked the approximate PAM "strength" based on the magnitude of CRISPRi knockdown of a Renilla luciferase target gene in M. smegmatis (Table 1). Numerous factors are likely to impact the efficacy of a given sgRNA (sgRNA target sequence composition, location of endogenous RNA polymerase pause sites, etc.), so PAM "strength" should be interpreted as a prediction but not a rule for CRISPRi knockdown efficiency. 3\. To achieve CRISPRi knockdown, identify PAM sequences in the template strand of your gene of interest (see Notes 1 and 2). Select a PAM with a desired approximate targeting strength (Table 1). 4\. To design the sgRNA oligos, extract the 21--24 nucleotides of DNA sequence immediately upstream of the PAM (see Note 3) such that the 5' nucleotide of the targeting sequence is an A or G. This is the first base that will be transcribed to synthesize the sgRNA, and A or G is strongly preferred as an initiating nucleotide in mycobacteria \[23\]. If there is no 5' A/G in this 21--24 nucleotide window, select 23 bases immediately upstream of the PAM and append a 5' G to the selected sequence. This 21--24 nucleotide sequence will be the sgRNA targeting sequence (see Notes 4 and 5 regarding off-target effects and "bad seed" sequences). 5\. Append a 5'-GGGA-3' sequence 5' to the sgRNA targeting sequence and order this as the top oligo for cloning the sgRNA. 6\. Take the reverse complement of the sgRNA targeting sequence and append a 5'-AAAC-3' sequence 5' to the reverse complemented sgRNA targeting sequence. This is the bottom oligo for cloning the sgRNA. 7\. When annealed, the top and bottom oligos will form a dsDNA fragment with sticky end overhangs (the bolded sequences in the examples below) for ligation into the CRISPRi backbone. As controls, we provide two pairs of oligos for cloning an sgRNA against rpoB (rv0667) in M. tuberculosis and an sgRNA against mmpL3 (MSMEG\_0250)in M. smegmatis. Successful CRISPRi targeting of rpoB and mmpL3 will result in a severe growth defect and serve as a positive control for CRISPRi implementation. rpoB\_rv0667 \_T: 5'-GGGAGACATCGTCGAAACGAGGGTC-3'. rpoB\_rv0667\_B: 5'-AAACGACCCTCGTTTCGACGATGTC-3'. mmpL3\_ms0250\_T: 5'-GGGAGCGACAGACTGGCTGCCCT CGTC-3'. mmpL3\_ms0250\_B: 5'-AAACGACGAGGGCAGCCAGTCTGT CGC-3'.   **Annealing sgRNA Oligos for Ligation into the CRISPRi Plasmid Backbone** The oligos can be annealed by heating the oligo mixture to 95 C, then slowly reducing the solution to ambient temperature to allow annealing of the oligos. 1\. Set up the oligo annealing reaction in PCR tubes as below: 46 μL oligo annealing buffer. 2 μL 100 μM top oligo. 2 μL 100 μM bottom oligo. 2\. Place the PCR tubes in a thermocycler and anneal the oligos using the following cycling conditions: 95 C for 2min. 0.1 C/s to 25 C. END. 3\. Dilute the annealed oligos 1:100 in deionized water. **BsmBI-Digestion of the CRISPRi Plasmid Backbone** 1\. Set up a BsmBI restriction digest as follows: 5 μL NEBuffer 3.1. 4 μL BsmBI-v2 (10,000 U/mL). 2--5 μg CRISPRi backbone (plJR965). Water up to 50 μL. 2\. Incubate at 55 C for 4 h---overnight. 3\. Cast a 0.7% (w/v) TAE agarose gel with a blue-light transilluminator compatible, DNA intercalating dye (such as SYBR Safe). 4\. Load the entire digestion reaction on the gel. As a control to assess efficient plasmid digestion, also load 100 ng of undigested plJR965, leaving at least one empty lane between the control and the digestion reaction. 5\. Run the DNA gel at 120 V for approximately 45 min. 6\. Use a blue-light transilluminator to visually confirm BsmBI digestion of the CRISPRi plasmid. The digested, linear plasmid should be approximately 8.6 kb and migrate at a higher molecular weight than the undigested, supercoiled plasmid. 7\. Gel extract the digested CRISPRi backbone using the QIA quick Gel Extraction Kit. **Ligation of the Annealed Oligos into the CRISPRi Backbone** The annealed sgRNA oligos will be cloned into the CRISPRi plasmid backbone using ligation of complementary "sticky end" overhangs. 1\. Each oligo pair will be ligated into the CRISPRi plasmid in separate ligation reactions. A control ligation with digested vector in the absence of oligos should also be prepared to confirm the complete digestion of the CRISPRi backbone. To prepare a master mix for the ligation reactions, multiply the number of reactions by the following required volumes of each reagent and aliquot the reagents into a master mix tube: 0.25 μL vector (BsmBI-digested, gel-purified; approximately 9 ng, see Subheading 3.3). 0.5 μL10 T4DNAligase buffer. 0.25 μL T4 DNA ligase (2,000,000 U/mL). 3.5 μL deionized water. 2\. Transfer 4.5 μL of the master mix to a tube with 0.5 μL annealed oligos (see Subheading 3.2). 3\. Incubate at room temperature for 30 min---overnight. 4\. Transform 0.5 μL of the ligation reaction into 5 μL of chemically competent E. coli cells. 5\. Add 50 μL of LB broth to the transformation and recover for 1 h at 37 C. 6\. Plate the transformation on LB with 50 μg/mL kanamycin to select for kanamycin-resistant clones. 7\. Miniprep a single clone from each reaction and validate cloning by Sanger sequencing with oligo: 5'-TTCCTGTGAAGAGCCATTGATAATG-3'. In our experience, sgRNA cloning in E. coli with this protocol typically yields a \>90% success rate. **Preparation of Electrocompetent Mycobacteria** 1\. Start a culture of M. tuberculosis in 5 mL of 7H9-OADC Tween80 at an OD600 of 0.025 in a T-25 flask. Grow in stationary conditions at 37 C with 5% CO2 for approximately 3--4 days to saturation (OD600 \~ 1.0). 2\. Expand the culture in a 125 mL screw cap Erlenmeyer flask by transferring 3 mL of the starter culture to 22 mL 7H9-OADC Tween80. Grow for at least 3 generations (approximately 3 days) to an OD600 of 0.8--1.0 at 37 C with shaking. 3\. Transfer the culture to a 50 mL conical tube. 4\. Centrifuge the cells in the 50 mL conical tube at 4000 g for 10 min. 5\. Discard the supernatant, and gently resuspend the pelleted cells in 1 mL 10% glycerol with a p1000 pipette. 6\. Add 24 mL of 10% glycerol to bring up to the final volume to 25 mL. 7\. Repeat steps 4--6 twice for a total of three washes. 8\. Resuspend the washed cells in 1 mL 10% glycerol. This is sufficient for at least 10 transformations. Competent cells should be used the same day. **Electroporation of CRISPRi Constructs into Electrocompetent Mycobacteria** 1\. Aliquot \~1 μL (at least 100 ng) of the CRISPRi construct (see Subheading 3.4, step 4) to the bottom of a labeled 2 mL screw cap microfuge tubes. 2\. Add 100 μL of electrocompetent M. tuberculosis to the CRIS PRi construct in the 2 mL screwcap tube. 3. Pipette up and down to mix and transfer to a 2 mm electroporation cuvette. 4\. Place the cuvette in the electroporation pod and electroporate the bacilli with the following settings: 2500 V, 700 Ω,25μF. 5\. Recover the cells by adding 1 mL of 7H9-OADC-Tween80 to the cuvette and transferring the cells to the 2 mL screw cap microfuge tube for recovery and outgrowth. 6\. Incubate the transformation recovery standing in a 37 C incubator overnight. 7\. Pellet the recovered cells by centrifugation in a microcentrifuge at 4000 g for 5 min. 8\. Discard the supernatant and resuspend the cell pellet in 100 μL of 7H9-OADC-Tween80. 9\. Plate cells on 7H10-OADC-Tween80 with 20 μg/mL kanamycin to select for transformants. If knockdown of your gene of interest is expected to produce a phenotype on agar plates (e.g., lack of growth), additional plating of half of the transformation on a separate kanamycin plate in the presence of 100 ng/mL anhydrotetracycline (ATc) can confirm the knock down phenotype (see Notes 6 and 7). **Quantification of Transcriptional Knockdown by qRT PCR** 1\. Grow a 5 mL starter culture of the desired M. tuberculosis CRISPRi strains, prepared as in Subheading 3.6. As a control, also include a nontargeting control strain containing the parent CRISPRi plasmid, plJR965. 2\. Grow in a T-25 flask in stationary conditions at 37 C with 5% CO2 in 7H9-OADC-Tween80 with 20 μg/mL kanamycin to saturation (OD600 \~ 1.0). 3\. Dilute the culture in a new T-25 flask to an OD600 of 0.05 in 5 mL of the same media. 4\. Grow for 3 days to log phase (OD600 0.4--0.8) in stationary conditions at 37 C with 5% CO2. 5\. Dilute the culture in a new T-25 flask to an OD600 of 0.1--0.2 in 5 mLofthesamemedia+100ng/mL ATc(seeNote6). ATc is used to induce the expression of Sth1 dCas9 and the sgRNA. GrowinaT-25flaskasabovefor 2--3generations (2--3 days) to allow for the expression of CRISPRi components and transcriptional silencing of the target gene. 6\. Transfer a culture volume containing 2 OD600unitsofcells (for example, 2 mL of a culture at OD600 of 1.0) or approximately 6 108cells to an equal volume of GTC buffer in a 15 mL conical (see Note 8). 7\. Pellet the cells by centrifugation at 4000 g for 10minat4C. 8\. Resuspend the pellet in 1 mL of TriZol with a p1000. Transfer the pellet to a Lysing Matrix B tube. 9\. Lyse the cells by bead beating at 6500 rpm in 3 1 min cycles, incubating on ice for 1 min between each cycle. 10\. Add 200 μL of chloroform to the lysed cells in TriZol. Vortex for 5 s. 11\. Allow the bead beating tubes to sit for 2 min to allow dissociation of nucleoprotein complexes (samples can be frozen at 80 C prior to RNA isolation if desired). 12\. Proceed with total RNA isolation and cDNA synthesis as per kit instructions. 13\. The dCas9-sgRNA complex functions as a roadblock for elongating RNA polymerase or a steric block when binding to the target gene promoter. To quantify target gene knockdown, quantify target gene mRNA levels downstream of the sgRNA target sequence binding site (see Notes 9--11). 14\. Set up qPCR reactions to determine transcript levels of the target gene and a housekeeping gene (e.g., sigA). Use the ΔΔCt method to determine the fold knockdown of your gene of interest by comparing transcript levels of your target gene relative to a housekeeping gene. The relevant comparisons can be your M. tuberculosis CRISPRi strain grown in the presence or absence of ATc, or the control M. tuberculosis CRISPRi strain containing plJR965 grown in the presence of ATc. **Design of CRISPRi-Resistant Complementation Constructs** To confirm that the phenotype seen in a M. tuberculosis CRISPRi strain is due to the transcriptional knockdown of the targeted gene, complementation with a CRISPRi-resistant allele of the target gene is recommended (see Note 12). Since dCas9 is guided by sequence complementarity of the target and sgRNA, a construct expressing a CRISPRi-resistant allele of the targeted gene can be generated by Gibson assembly to introduce silent mutations in the sgRNA bind ing site and/or the PAM. 1\. To disrupt CRISPRi targeting, introduce at least 2 mutations in the target sequence and/or the PAM without affecting the amino acid sequence of the ORF. Mutate the PAM to a sequence not recognized by Sth1 dCas9 if possible, or to a weaker PAMif the available silent mutations do not completely inactivate the PAM (refer to Table 1). In the sgRNA target sequence, silent mutations should be made in the seed sequence, or the 8--9 most PAM-proximal nucleotides \[12, 24\]. 2\. Use the NE Builder tool(https://nebuilder.neb.com) to design a Gibson assembly cloning scheme and oligos to amplify and clone the targeted gene into the desired expression vector (see Notes 13 and 14). To introduce the silent, CRISPRi-abrogating mutations, design an oligo containing the region of silent mutations to be made, and 20 bp flanking upstream and downstream of the region to be mutated. Also design the reverse complement of that oligo. 3\. Using the PCR cloning scheme designed by NE Builder, PCR amplify the targeted gene in 2 fragments using the NE Builder designed oligos and the oligos to introduce silent mutations (see Note 15). 4\. Use Gibson assembly to clone the CRISPRi-resistant expression construct into the desired expression vector. In parallel, also clone a CRISPRi-sensitive (i.e., wild-type) allele into the expression construct (see Note 16). 5\. Sanger sequence a clone of each construct to confirm proper assembly and/or introduction of the silent mutations. 6\. Transform the CRISPRi-resistant and CRISPRi-sensitive con structs into the relevant electrocompetent cells of the M. tuberculosis CRISPRi strain. 7\. Perform phenotypic assays such as growth and survival assays and microscopy to confirm complementation of the CRISPRi knockdown phenotypes. **Targeting Multiple Genes with CRISPRi** To clone multiple sgRNAs into the same plasmid, the CRISPRi backbones were designed with a SapI-based Golden Gate cloning site. This site is placed downstream of the first sgRNA scaffold. In principle, the SapI-based Golden Gate approach allows for the cloning of an additional seven sgRNA cassettes in tandem into a single CRISPRi backbone, which would in turn direct the expression of a total of eight sgRNAs from a single plasmid. 1\. Clone all individual sgRNAs into the CRISPRi backbone (see Subheadings 3.1--3.4 for detailed design and cloning instructions). 2\. Amplify each \[promoter-sgRNA-terminator\] cassette with primers that contain SapI-restriction sites as well as compatible DNA overhangs with the SapI-based Golden Gate handle (Fig. 4, see Table 2 for a list of primers to clone up to 7 additional sgRNAs). 3\. Gel purify the PCR amplified \[promoter-sgRNA-terminator\] cassettes. 4\. Perform Golden Gate cloning, which consists of one-pot restriction digestion and ligation (see Note 17). Set up the Golden Gate reaction as follows: 2 μL DTT(10 mM). 2 μL ATP (10 mM). 2 μL10 CutSmart Buffer. 0.5 μL T4 DNA ligase (2,000,000 U/mL). 1 μL SapI (10,000 U/μL). 20 fmol CRISPRi vector (\~100 ng). 40 fmol each \[promoter-sgRNA-terminator\] cassette. Water up to 20 μL. 5\. Incubate at 37 Cfor1h, then65Cfor20min(seeNote18). 6\. Transform 0.5 μL of the Golden Gate reaction into 5 μL of commercially available, chemically competent E. coli cells (see Note 19) 7\. Add 50 μL of LB broth to the transformation and recover for 1 h at 37 C. 8\. Plate the transformation on LB with 50 μg/mL kanamycin to select for kanamycin-resistant clones. 9\. Miniprep and validate cloning by Sanger sequencing with the following primers: GG\_seq\_1: 5'-TGCGGCGCTTTTTTTTTTGAATTC-3'. GG\_seq\_2: 5'-CTGCGTTATCCCCTGATTCTG-3'. In larger arrays of multiple sgRNA cassettes, the oligos used to clone each sgRNA target sequence, as described in Subheading 3.1, can be used in Sanger sequencing for cloning validation.  **CRISPRi Transcriptional Interference in M. smegmatis** 1\. All culturing of M. smegmatis is done with 7H9-ADC Tween80 for liquid media or 7H10-ADC-Tween80 for solid media (see Note 20). 2\. To prepare competent cells, grow M. smegmatis overnight to saturation in 5 mL of 7H9-ADC-Tween80 in a 30 mL inkwell shaking at 37 C. In a 125 mL inkwell, dilute the overnight culture to an OD600of0.01--0.02inatotalvolumeof25mLof the same media. Grow for 16 h to an OD600 of \~1.0. Prepare electrocompetent cells as described for M. tuberculosis (see Sub heading 3.5). 3\. Electroporation's can be performed in M. smegmatis as in M. tuberculosis (see Subheading 3.6). Transformations should be recovered in 7H9-ADC-Tween80 for at least 3 h prior to plating on 7H10-ADC-Tween80 with 20 μg/mL kanamycin. 4\. The level of target-gene transcriptional repression can be quantified similarly in M. smegmatis as in M. tuberculosis. Grow an M. smegmatis CRISPRi strain to saturation (OD600 \~ 1.0) shaking at 37 C in 7H9-ADC-Tween80 with 20 μg/mL kanamycin in a 30 mL inkwell. 5\. Dilute the culture in at least 5 mL 7H9-ADC-Tween80 with 20 μg/mL kanamycin in a new 30 mL inkwell for at least 3 generations (9 h) at 37 C to log phase (OD600 0.4--0.8). 6\. Dilute the culture in at least 5 mL 7H9-ADC-Tween80 in a new 30 mL inkwell with 20 μg/mL kanamycin and 100 ng/ mLATc to an OD600 of 0.1. 7\. Grow for at least 3 generations at 37 C in shaking conditions to allow for the expression of CRISPRi components and to allow for dCas9:sgRNA-mediated transcriptional interference at the gene of interest. 8. Harvest cells for RNA extraction and cDNA preparation as with M. tuberculosis RNA samples. **NOTES** 1\. Previous reports suggested CRISPRi is more efficient when targeting the 5', rather than 3', end of the ORF. We and others have not observed such position-dependent differences in CRISPRi efficacy \[9, 20, 21\]. 2\. Evidence from a recent genome-wide CRISPRi screen in E. coli suggests that stronger transcriptional interference is achieved, on average, by targeting the gene ORF rather than by targeting the gene promoter \[20\]. However, this is not a consensus viewpoint in the field \[22\]. 3\. The canonical SpyCas9 has an optimum sgRNA targeting sequence length of 20 nt. By contrast, unpublished results from our lab suggest Sth1Cas9 appears to have an optimum sgRNA length of 21--24 nt. 4\. Off-target effects have been reported in bacterial CRISPRi systems \[21\]. In the SpydCas9 CRISPRi system used in E. coli, Cui and colleagues found that sgRNAs with as few as 9 nucleotides of perfect complementarity between the PAM-proximal region of the sgRNA targeting sequence (i.e., the seed sequence) and a PAM-containing off-target site could mediate off-target gene repression \[21\].Given the prevalence of the SpyCas9 PAM (5'-NGG-3') in the bacterial chromo some, off-target gene repression was common enough to war rant consideration in sgRNA design. The authors recommend checking a selected sgRNA for off-target effects by identifying perfect matches to the 9 nucleotide sgRNA seed region. If there are multiple perfect matches to the bacterial chromosome, and these potential off-target sites have a PAM and target the non-template strand within an ORF, or either strand in a promoter, an alternatives gRNA should be designed. Note that the more restrictive PAM for Sth1Cas9 means that off-target effects are likely to be rarer than those observed with SpyCas9. Potential off-target effects for Sth1 sgRNAs can be identified as described above. 5\. Cui et al. also observed a phenomenon they describe as the "bad seed" \[21\]. Using the Spy dCas9 CRISPRi system, "bad seeds" were defined as specific 5 nucleotide sequences in the sgRNA seed sequence that result in target-independent fitness costs. The mechanism responsible for this phenomenon has not been identified, but the bad-seed effect could be alleviated by reducing Spy dCas9 expression levels. 6\. ATc is light sensitive. Cultures should be kept in the dark with minimal light exposure. Flasks and plates can be wrapped in aluminum foil to minimize light exposure. 7\. Transformations of CRISPRi plasmids encoding sgRNAs that achieve strong inhibition of in vitro essential genes plated in the presence of ATc should result in growth inhibition. It is normal for some "escaper" colonies to arise in the presence of ATc at a low frequency (\~1:500 to 1:1000 transformants, unpublished data). 8\. GTC buffer contains: guanidine thiocyanate to lyse cells and inhibit RNases; N-lauryl sarcosine to inhibit new rounds of transcription initiation during cell lysis; and sodium citrate to chelate divalent cations that can contribute to RNA hydrolysis. 9\. Another method for assessing CRISPRi target gene knock down is by monitoring the targeted gene's protein levels by western blot. However, this is not as broadly applicable given the paucity of antibodies to endogenous M. tuberculosis proteins. 10\. We use the Primer Quest tool from IDT (https://www.idtdna. com/Primer Quest/Home/Index) to design 2--3 qPCR primer pairs for the target gene of interest. Each qPCR primer pair is then validated for efficiency, specificity, and the linear range of amplification using standard qPCR approaches. A single amplification product is confirmed for each validated qPCR primer pair by inspection of the amplification melt curve. 11\. Due to the common occurrence of promoters in the M. tuberculosis genome \[25, 26\], including within ORFs and in the sense and antisense directions, exercise caution when interpreting the magnitude of target knockdown by qRT-PCR. For instance, CRISPRi knockdown of some target genes may appear ineffective by qRT-PCR. One possible explanation for this lack of measured knockdown is the presence of multiple promoters driving transcription through the target gene, not all of which are disrupted by the chosen sgRNA. In such instances, consult published transcription start site (TSS) annotations in the M. tuberculosis genome to determine the location and direction of TSSs proximal to the gene of interest. Design and validate qPCR amplicons that avoid quantifying interfering transcripts and/or use strand-specific qPCR to measure direction-specific transcription. 12\. It is important to consider the operon structure of genes targeted with CRISPRi. CRISPRi targeting of a gene in an operon results in transcriptional knockdown of the targeted gene and all downstream genes in the operon. This polar effect is due to the inhibition of RNA polymerase elongation beyond the site of dCas9 occupancy, as defined by the sgRNA targeting sequence. Hence, genes downstream of the sgRNA targeting sequence will also experience CRISPRi-mediated transcriptional repression. When targeting operonic genes with CRISPRi, it is therefore important to consider whether the genes downstream of the targeted gene could be contributing to or the source of the phenotype of interest. Peters and colleagues reported polar effects on upstream genes \[19\], where genes upstream of the CRISPRi-targeted gene were also repressed. We and others have not observed this "upstream" polar effect \[9, 21\] 13\. If not supplied in the expression vector, the promoter driving expression of the complementation allele can be selected by the user. In NE Builder, add the promoter sequence as an additional fragment between the expression vector backbone and 5' end of the first gene fragment. PCR amplify the promoter sequence and clone into the expression vector as per NE Builder instructions. 14\. In order for this vector to be compatible with the CRISPRi backbone, ensure that the expression vector does not rely on kanamycin resistance for positive selection or the L5 integrase for chromosomal integration. 15\. The first amplified fragment should contain an upstream overhang with sequence identity to one end of the linearized expression vector, and a downstream overhang with the silent mutations and sequence identity to the upstream end of the second fragment. The second amplified fragment should contain an upstream overhang with the silent mutations and sequence identity to the downstream end of the previous fragment, and a downstream overhang with sequence identity to the other end of the linearized expression vector. 16\. Lack of complementation by expression of a CRISPRi-sensitive allele confirms that complementation of the CRISPRi knock down phenotype by a CRISPRi-resistant allele is not due to the expression of a second allele of the targeted gene, but rather due to the specific expression of a CRISPRi-resistant allele of the targeted gene. 17\. The Golden Gate reaction will digest the desired parent sgRNA plasmid (i.e., the CRISPRi plasmid that contains your first sgRNA at the BsmBI-based cloning site) and the PCR-amplified and gel-purified sgRNA cassettes with SapI and simultaneously ligate with T4 DNA ligase. If there are difficulties with cloning efficiency using the described Golden Gate approach, the restriction digests, gel purification, and ligations can be performed in sequential steps to facilitate troubleshooting. 18\. These thermocycler conditions are suggested for up to 5 inserts. For 6 or more inserts, cycle 30 times between 37 C for 1 min and 16 C for 1 min, then heat at 60 C for 5 min. 19\. When cloning multiple sgRNA cassettes into a single Golden Gate site (i.e., such that a single plasmid will express more than 2 sgRNAs), it may be necessary to increase the volumes of competent E. coli cells used to increase cloning yield. 20\. The methods described in this protocol are likely applicable to other mycobacterial species not discussed here, including M. abscessus, M. avium complex, M. marinum, and others.