Programmable Gene Knockdown in Diverse Bacteria Using Mobile‐CRISPRi

Abstract Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene‐knockdown tool that uses an RNA‐protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile‐CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile‐CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile‐CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile‐CRISPRi vectors, Tn7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria, and ICEBs1 transfer of Mobile‐CRISPRi to Bacillales. © 2020 The Authors. Basic Protocol 1: sgRNA design Basic Protocol 2: Cloning of new sgRNA spacers into Mobile‐CRISPRi vectors Basic Protocol 3: Tn7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria Basic Protocol 4: ICEBs1 transfer of Mobile‐CRISPRi to Bacillales Support Protocol 1: Quantification of CRISPRi repression using fluorescent reporters Support Protocol 2: Testing for gene essentiality using CRISPRi spot assays on plates Support Protocol 3: Transformation of E. coli by electroporation Support Protocol 4: Transformation of CaCl2‐competent E. coli


INTRODUCTION
CRISPRi (Fig. 1A) is a contemporary gene-perturbation strategy with substantial advantages over classic genetic approaches (e.g., gene deletions, transposon mutagenesis). First, CRISPRi is programmable (Qi et al., 2013): to specify a gene for knockdown, one need only alter the first 20 nucleotides (nt) of the sgRNA (known as the "spacer") to match the target gene (provided that there is a nearby protospacer-adjacent motif, or PAM; see Strategic Planning). This programmability makes it straightforward to construct

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Current Protocols in Microbiology single-gene knockdowns or knockdown libraries targeting defined sets of genes (Liu et al., 2017;Peters et al., 2016) or all genes in the genome (Lee et al., 2019;Rousset et al., 2018;Wang et al., 2018;Yao et al., 2020). Because the sgRNA spacer uniquely identifies the target gene, spacers can act as barcodes to obtain counts of individual knockdown strains and measure their fitness in pooled growth experiments. Second, CRISPRi is inducible and titratable, and therefore can be used to target essential genes (Li et al., 2016;Peters et al., 2016). The timing and extent of CRISPRi knockdown can be controlled by expressing the sgRNA and dcas9 genes from inducible promoters. Further, knockdown gradients can be achieved by expressing CRISPRi components from constitutive promoters of differing strengths , by using truncated spacers (Vigouroux, Oldewurtel, Cui, Bikard, & Teeffelen, 2018), or by systematically mutating the sgRNA to introduce mismatches between the spacer and target gene that reduce knockdown efficacy to a predictable extent (Hawkins et al., 2020). Finally, CRISPRi can be multiplexed to target several genes in the same cell by cloning arrays of sgRNAs with different spacers (Ellis, Kim, & Machner, 2020;Peters et al., 2016;Reis et al., 2019). These advantages suggest that CRISPRi will become a common tool for bacterial functional genomics in the near future.
Despite the utility of CRISPRi, its widespread adoption in bacteria has been limited by a lack of generalizable systems. To facilitate the use of CRISPRi in diverse bacteria, we developed Mobile-CRISPRi-a suite of modular vectors that transfer by conjugation and stably integrate into the genomes of recipient bacteria (Peters et al., 2019). All Mobile-CRISPRi vectors contain (1) dcas9, (2) an sgRNA with either a targeting spacer or restriction sites for cloning new spacers, and (3) an antibiotic resistance marker for selection in recipient bacteria (Fig. 1B). The modularity of Mobile-CRISPRi enables facile cloning of sgRNA libraries. There are two general sets of Mobile-CRISPRi vectors: ICEBs1-based vectors for Gram-positive bacteria related to Bacillus subtilis (i.e., order Bacillales) that integrate downstream of the leu2 tRNA (Fig. 1C), and Tn7-based vectors for Gram-negative bacteria that integrate downstream of the glmS gene (Fig. 1D). Tn7 Mobile-CRISPRi vectors transfer from a diaminopimelic acid (DAP)-dependent donor strain of Escherichia coli to recipient strains of interest via the RP4 conjugation machinery and require a helper plasmid for integration (also transferred from E. coli) that expresses the Tn7 transposition genes (tnsABCD; triparental mating). ICEBs1 Mobile-CRISPRi vectors are first integrated into the genome copy of ICEBs1 in a B. subtilis donor strain using natural competence transformation and are then transferred to recipient strains of interest through induction of the ICEBs1 conjugation machinery (biparental mating). Integration of Mobile-CRISPRi downstream of glmS or leu2 does not impart a fitness defect in the strains tested (although Tn7 insertion might not be neutral in all species, and expression of dcas9 may be toxic in some bacteria; see Commentary).
Mobile-CRISPRi transfer, integration, and knockdown efficiencies vary by recipient strain for reasons that are sometimes but not always understood (Banta, Enright, Siletti, & Peters, 2020;Peters et al., 2019;Qu et al., 2019). To rapidly assess Mobile-CRISPRi knockdown efficacy, we developed "test" vectors for both the Tn7 and ICEBs1 systems that contain either an mRFP or an sfGFP gene (encoding monomeric red fluorescent protein or superfolder green fluorescent protein, respectively) and an sgRNA targeting mRFP or sfGFP that allows knockdown to be easily measured in live cells using a fluorometer or flow cytometer. The modular nature of Mobile-CRISPRi enables straightforward knockdown optimization approaches, such as replacing the sgRNA and dcas9 promoters or dcas9 ribosome-binding site with native regulatory sequences from the recipient bacterium (see Commentary  [Peters et al., 2019]; α-Proteobacteria: Zymomonas mobilis [Banta et al., 2020]) and Gram-positive species (Firmicutes: B. subtilis, Listeria monocytogenes, Staphylococcus aureus, and Enterococcus faecalis [Peters et al., 2019]).
Here, we describe the following basic protocols: (1) sgRNA design for selecting and computationally optimizing guide spacers (Basic Protocol 1), (2) cloning of new sgRNA spacers into Mobile-CRISPRi vectors to target specific genes for knockdown (Basic Protocol 2), (3) Tn7 transfer of Mobile-CRISPRi to Gram-negative bacteria via conjugation with E. coli donors (Basic Protocol 3), and (4) ICEBs1 transfer of Mobile-CRISPRi to Bacillales via conjugation with a B. subtilis donor (Basic Protocol 4). We also provide the following support protocols: (1) quantification of CRISPRi repression using fluorescent reporters that measure knockdown in live cells (Support Protocol 1), (2) testing for gene essentiality using CRISPRi spot assays on plates to examine the effects of CRISPRi gene knockdown on plating efficiency (Support Protocol 2), (3) transformation of E. coli by electroporation (Support Protocol 3), and (4) transformation of CaCl 2 -competent E. coli to facilitate cloning sgRNA spacers and create donor strains for Mobile-CRISPRi mating (Support Protocol 4).

Biosafety caution
CAUTION: Follow all biosafety requirements relevant to the microorganism under investigation. See Burnett et al. (2009) for more information.

STRATEGIC PLANNING
Before beginning CRISPRi experiments in a new strain, one must first design sgRNA spacers against target genes (see Basic Protocol 1). In general, desirable sgRNAs are on target (have only one binding site in the genome) and have high efficacy (although lower-efficacy guides may be useful for targeting essential genes; Hawkins et al., 2020;Vigouroux et al., 2018). To design on-target guides, we use a strategy developed by the Weissman and Gross labs that takes into account the DNA-binding preferences of the dCas9-sgRNA complex to score guide specificity (Gilbert et al., 2014;Peters et al., 2019). For instance, noncomplementarity between the sgRNA and a target site proximal to the PAM (the sgRNA "seed" region) has a strong negative effect on binding, whereas PAMdistal mismatches (e.g., 15-20 nt away from the PAM) have less impact on binding and may result in off-target effects. To identify and eliminate off-target guides, we strongly recommend designing sgRNA spacers for the entire genome at once (see Basic Protocol 1). If no closed genome sequence exists for the strain of interest, it is not possible to predict off-target sgRNA activity; therefore, gene-knockdown phenotypes must be confirmed by multiple guides. Because bacterial genomes are relatively small, most sgRNA spacers have high specificity (i.e., a maximum specificity score of 39 in our code below). sgRNA knockdown efficacy is complex and the subject of ongoing research (Calvo-Villamañán et al., 2020), although some general rules have emerged. The clearest rule is that guides targeting the nontemplate strand of the gene (antisense, or "anti" in our code below) have much higher efficacy than template-targeting guides (Bikard et al., 2013;Qi et al., 2013); the mechanistic underpinnings of this effect are unknown. Early observations suggested that sgRNAs targeting the 5 ends of genes are more efficacious, but subsequent studies have shown that guide efficacy is constant across genes on average Rousset et al., 2018). Nonetheless, we tend to prefer spacers that target toward the 5 ends of genes because the effects of the CRISPRi transcription block on translation are unclear. Targeting promoters with CRISPRi can also be effective, but we prefer to target genes because of the lack of promoter location data in the majority of bacteria and the fact that spacer distribution is more limited in intergenic regions (i.e., there are fewer NGG PAM sequences in AT-rich promoter regions).

CLONING NEW sgRNA SPACERS INTO MOBILE-CRISPRi VECTORS
This protocol describes the construction of Mobile-CRISPRi vectors encoding an sgRNA spacer targeting a gene of interest. Two oligonucleotides are designed such that when annealed, they form the desired sgRNA spacer sequence with overhangs enabling ligation into a BsaI-digested Mobile-CRISPRi plasmid (Fig. 2). The resulting plasmid, which contains the entire Mobile-CRISPRi system encoding the sgRNA, dCas9, and antibiotic resistance marker between Tn7 transposon ends, can be used as a donor for transposition of the system into the recipient att Tn7 site. Figure 2 sgRNA spacer cloning. Shown here is the sgRNA module from the Mobile-CRISPRi plasmid pJMP1339. Annealed oligos with BsaI-compatible sticky ends are ligated into the BsaI-cut vector (BsaI recognition sites are lost in the cloning process). This figure depicts a spacer targeting mRFP, but 20-nt spacer sequences targeting any gene of interest can be cloned using this protocol.

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Current Protocols in Microbiology
14. Pellet cells from entire 5-ml culture and extract plasmid DNA using a kit following the manufacturer's instructions.
The Mobile-CRISPRi plasmids are fairly large (∼10-12 kb) and low copy. Extracting the entire 5-ml culture and eluting in ∼40 μl typically yields ∼100 ng/μl plasmid DNA. Warming the elution buffer to 50°C and waiting ∼2 min after applying the buffer to the column before spinning may increase yield.
15. Sequence the region of the plasmid near the insertion site to confirm the insertion and the fidelity of the cloning.

Tn7 TRANSFER OF MOBILE-CRISPRi TO GRAM-NEGATIVE BACTERIA
This protocol transfers the Tn7-based Mobile-CRISPRi system into the chromosome of a Gram-negative bacterium of interest. E. coli donor strains have a chromosomal copy of the RP4 transfer machinery to mobilize the Tn7 plasmids. A plasmid with a Tn7 transposon carrying CRISPRi components and a second plasmid encoding Tn7 transposition genes are transferred to recipient bacteria by triparental mating. In the recipient, transposition proteins integrate the CRISPRi system into the recipient genome Banta et al.

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Current Protocols in Microbiology LB + DAP E. coli pir + "mating strain" Blodgett et al. (2007) downstream of the glmS gene. Selection on plates lacking DAP eliminates the DAPdependent E. coli donors, whereas R6K ori plasmids are lost because they cannot replicate in recipient cells that lack the pir gene. Antibiotic selection results in retention of only the recipients with an integrated CRISPRi system. Once integrated into the chromosome, the Mobile-CRISPRi system is stable without further antibiotic selection in all organisms tested so far. This method has been used with a variety of recipient γ-Proteobacteria, including Acinetobacter, Enterobacter, Escherichia, Klebsiella, Proteus, Pseudomonas, Salmonella, Shewanella, and Vibrio, as well as the α-Proteobacterium Zymomonas, and is likely to be adaptable to a wider range of bacteria.

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Current Protocols in Microbiology 9. Using ethanol-sterilized tweezers, transfer the filter to a microcentrifuge tube containing 200 μl 1× PBS. Vortex ∼15 s to dislodge cells from the filter and resuspend in the buffer. 11. Stock strains in medium + 15% glycerol and store at −80°C.
12. Once recipient strains have been generated, CRISPRi knockdown can be tested by targeting fluorescent proteins (Support Protocol 1) or by observing reduced plating efficiency upon targeting of essential genes (Support Protocol 2).

ICEBs1 TRANSFER OF MOBILE-CRISPRi TO BACILLALES
This protocol describes how to integrate the Mobile-CRISPRi system into the chromosome of a bacterium of interest using the B. subtilis integrative and conjugative element (ICEBs1, or conjugative transposon; Table 3). This protocol has been used with members of the Bacillales Firmicutes (e.g., Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis).

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Current Protocols in Microbiology 3. Mix 120 μl culture with ≥100 ng (∼1-5 μl) plasmid DNA in a deep 96-well plate, cover with breathable film, and incubate 10 min at 37°C without shaking, then 2 hr with shaking (900 rpm) on a microplate shaker.
4. Plate cells on LB agar + 7.5 μg/ml kanamycin and incubate 16-24 hr at 37°C. Plate several amount to obtain isolated colonies and/or restreak for isolation on a new selection plate.
Do not select for chloramphenicol resistance at this step; the kanamycin marked Mobile-CRISPRi cassette has replaced the existing ICEBs1 marker.
Do not store B. subtilis plates at 4°C, as B. subtilis loses viability at this temperature.

ICEBs1 transfer of Mobile-CRISPRi from B. subtilis donor to recipient strains
6. Inoculate 3 ml LB + 3.75 μg/ml kanamycin with a single colony of B. subtilis ICEBs1-CRISPRi donor strain and 3 ml LB (or strain-specific rich medium) with a single colony of the recipient strain, and incubate until exponential phase (∼2 hr) at 37°C with aeration (shaking at 250 rpm or rotating on a drum roller).
Adjust the medium and growth temperature of the recipient strain as necessary in this and subsequent steps. Table 3 for strain-specific media and antibiotic concentrations.

Use half the regular kanamycin concentration (3.75 μg/ml) for B. subtilis donor strains grown in liquid medium.
7. Dilute the starter cultures to OD 600 = 0.02 in 5 ml LB + 3.75 μg/ml kanamycin for donors or 15 ml LB (or recipient-specific rich medium) for recipients, and incubate until OD 600 ∼0.2 (∼1 hr) at 37°C with aeration.
Adjust volume of recipient culture depending on number of donor cultures (start ∼15 ml culture for each 4 donors).
8. Add 5 μl 1 M IPTG to 5 ml donor cultures (1 mM IPTG) to induce rapI expression and continue to incubate all cultures 1 hr at 37°C with aeration.

Expression of the ICEBs1 anti-repressor RapI induces conjugation genes found on the ICE element and promotes excision of ICEBs1 from the chromosome.
9. Adjust OD 600 of cultures to 0.9. For each mating, add 2.5 ml each of donor and recipient cultures to 5 ml Spizizen's medium in a 50-ml conical tube and vortex to mix.

Also set up control matings with no recipient (donor only) or no donor (recipient only).
10. Vacuum cell suspension through an analytical filter funnel to collect the cells on a CN filter, add 5 ml Spizizen's medium, and vacuum again to wash the filter.
11. Using flame-sterilized forceps, transfer the filter to a Spizizen's medium agar plate and incubate at 37°C for 3 hr.
Adjust mating time if necessary, depending on recipient.
12. Transfer each filter to a 50-ml conical tube containing 5 ml Spizizen's medium, and vortex to resuspend cells.
Adjust volume plated to obtain isolated colonies and/or restreak for isolation on a new selection plate.
15. Once recipient strains have been generated, CRISPRi knockdown can be tested by targeting fluorescent proteins (Support Protocol 1) or by observing reduced plating efficiency upon targeting essential genes (Support Protocol 2).

Construct strains with a fluorescent reporter to test CRISPRi knockdown
1. Transfer CRISPRi system with fluorescent reporter (see Table 1) to recipient strain according to Basic Protocol 3 (Tn7 transfer to Gram-negative bacteria) or 4 (ICEBs1 transfer to Bacillales).
You will need at least three strains: one encoding the fluorescent protein and an sgRNA targeting the fluorescent protein encoding gene, another encoding the fluorescent protein and a nontargeting sgRNA, and a third, nonfluorescent strain.

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Current Protocols in Microbiology 3. Serially dilute cultures 1:10,000 into 300 μl medium using a multichannel pipet as follows: dilute cultures 1:100 into LB (or appropriate medium), then dilute 1:100 again into LB (or appropriate medium) containing 0 or 1 mM IPTG (for CRISPRi-Tn7 version) or 0 or 0. 6. Analyze knockdown efficacy by first calculating fluorescence/OD 600 and then calculating a ratio of value of strains with and without the targeting sgRNA.
Be sure to subtract uninoculated medium background measurement from OD 600 measurement and nonfluorescent strain background measurement from fluorescent strain measurements.

TESTING GENE ESSENTIALITY USING CRISPRi SPOT ASSAYS ON PLATES
This protocol describes how to test for gene essentiality using CRISPRi. Serial dilutions of strains with Mobile-CRISPRi-encoding sgRNAs targeting genes of interest are spotted on agar plates with various concentrations of inducer. This protocol is useful when initially determining essentiality and for optimizing level of induction of Mobile-CRISPRi needed for partial knockdown for subsequent experiments.
2. Add 300 μl LB (or appropriate medium) to wells in a row of a deep 96-well plate. Inoculate wells with a single colony. Cover plate with sterile breathable film and incubate ∼16 hr at 37°C (or appropriate temperature), shaking at 900 rpm on a microplate shaker. 6. Image and analyze culture growth on plates. Reduced growth compared to the control (nontargeting sgRNA) will indicate reduced fitness of strains in which CRISPRi targets essential genes. Using plates with a range of IPTG concentrations may aid in selecting an inducer concentration appropriate for partial or complete knockdown.

TRANSFORMATION OF E. coli BY ELECTROPORATION
This protocol details the preparation of electrocompetent E. coli cells for transformation as well as the electroporation procedure. While either electrocompetent or chemically competent cells can be used for the protocols detailed here, electroporation is generally of higher efficiency and faster for small-scale experiments, but requires specialized equipment and is salt sensitive. In this protocol, electroporation is appropriate for transforming ligations into a cloning strain.

Include 300 μM DAP in plates and liquid medium if growing a DAP-dependent strain.
2. Place 5 ml LB medium in a culture tube, inoculate with a single colony, and incubate ∼14-18 hr at 37°C with aeration (shaking at 250 rpm or on a drum roller).
Do not overgrow culture. While culture is growing, prechill centrifuge and rotor, and label and prechill centrifuge bottles, tubes, and solutions. Perform remaining steps quickly and keeping cells chilled/on ice.
4. Swirl flask in a pan with an ice/water slurry for ∼5 min to quickly cool the culture.
In this and subsequent steps, cells can be centrifuged between 3000 and 6500 × g depending on centrifuge. Adjust time to minimize centrifugation time but ensure pellet formation.
6. Pour off supernatant and, while holding bottle in ice, resuspend cells in 2 ml 5% glycerol and then add 250 ml 5% glycerol, mix gently by inversion, and pellet the cells by centrifuging 10 min at 4000 × g, 4°C.
7. Pour off supernatant, resuspend cells in 2 ml 5% glycerol and then add 125 ml 5% glycerol, mix gently by inversion, and pellet the cells by centrifuging 10 min at 4000 × g, 4°C.
8. Pour off supernatant, resuspend cells in 5 ml 15% glycerol, transfer all 10 ml to a single 15-ml centrifuge tube, and pellet the cells by centrifuging 10 min at 4000 × g, 4°C.
Final resuspension volume can be adjusted from ∼1-5 ml/500 ml starting culture based on the number of cells needed per 50-μl volume per electroporation.
Distribute into single-use aliquots. Do not refreeze unused cells once thawed, but rather adjust aliquot size based on projected needs.

Transformation of E. coli by electroporation
10. Thaw electrocompetent E. coli cells ∼5 min on ice.
11. Prewarm two or three selective plates (i.e., LB agar + appropriate antibiotics and additives) for each transformation to 37°C.
13. Add up to 1 μl ligation or purified plasmid DNA to cells and transfer to a 0.1-cm-gap electroporation cuvette on ice.
Avoid introducing air bubbles into the suspension, which can result in an electrical discharge that reduces cell viability. It can be effective to set your pipet for several microliters less than the actual volume when transferring. If a bubble may have been introduced, tap cuvette on the counter several times. Ligation reactions contain salt, which may cause arcing if >1 μl of unpurified ligation is electroporated.

Follow the manufacturer's instructions for electroporation of E. coli for your electroporator.
15. Wipe cuvette dry with a Kimwipe, place in the holder, and then push the pulse button.
16. After completion of pulse, remove cuvette from the holder, add 800 μl SOC medium to the cuvette, mix by pipetting, and transfer cells and medium to a culture tube.
LB medium can also be used for outgrowth but may reduce transformation efficiency. Prewarming medium to 37°C and/or adding medium to cells as quickly as possible after the pulse may increase efficiency of transformation.
17. Incubate cultures 1 hr at 37°C shaking at 250 rpm or on a roller drum.
18. Plate transformation on two or three prewarmed selective plates to obtain isolated colonies. Incubate plates ∼16 hr at 37°C before proceeding with the rest of your experiment.
The amount to plate will depend on the competency of the cells. If the transformation efficiency is not known, plate several amounts (e.g., 200, 20, and 2 μl). If isolated colonies are not obtained, restreak for isolation on new plates. Alternatively, plate 80 μl of the transformation and then pellet the remaining cells (∼2 min at ∼6000 × g), spot on a plate, and then streak from that spot to obtain isolated colonies.

TRANSFORMATION OF CaCl 2 -COMPETENT E. coli
This protocol details the preparation of CaCl 2 -competent E. coli cells for transformation as well as the heat-shock procedure for transforming these cells. This method requires less specialized equipment than transformation by electroporation and is of lower efficiency but can easily be adapted to be higher throughput when many strains need to be constructed at once, such as when transferring intact plasmids to a mating strain.

Preparation of chemically competent E. coli cells
1. Streak strains onto LB agar plates from −80°C stocks to obtain isolated colonies. Incubate ∼14-18 hr at 37°C.
2. Place 5 ml LB medium in a sterile culture tube, inoculate with a single colony, and incubate ∼14-18 hr at 37°C with aeration (shaking at 250 rpm or on a drum roller).
Adjust size of culture as needed. 100 ml culture will prepare enough competent cells for two 96-well plates with 35 μl cells/well.
Do not overgrow culture. While culture is growing, prechill centrifuge and rotor, and label and prechill centrifuge bottles, tubes, and solutions. 4. Swirl flask in a pan with an ice/water slurry ∼5 min to quickly cool the culture.
In this and subsequent steps, cells can be centrifuged between 3000 and 6500 × g depending on the centrifuge. Adjust time to minimize centrifugation time but ensure pellet formation.
6. Pour off supernatant and, while holding tubes in ice, resuspend cells in 25 ml 50 mM CaCl 2 /10 mM Tris, pH 7.5. Mix gently by inversion and place on ice for 15 min.
8. Pour off supernatant and, while holding tubes in ice, resuspend cells from each tube in 3.3 ml 50 mM CaCl 2 /10 mM Tris, pH 7.5/15% glycerol, combine resuspended cells into one tube, and place on ice for 30 min. 11. Add 1-2 μl plasmid DNA (>10 ng) to cells and gently pipet to mix. Close strip caps or cover 96-well plate with adhesive foil.
12. Hold on ice for 30 min, incubate for exactly 2 min in a 42°C heat block, and then hold on ice for 5 min.
Banta et al.

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Current Protocols in Microbiology   1 M MgCl 2 (see recipe) and 3.6 ml sterile 20% (w/v) glucose (see recipe) and stir to mix. Aliquot 10 ml/tube and store between −20°C and 25°C.

Spizizen's medium and agar plates
Combine all components listed in Table 7 and filter sterilize.

Background Information
CRISPR interference (CRISPRi) is a programmable gene-knockdown tool based on the clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune systems found in bacteria and archaea that restrict viral and plasmid DNA and RNA. Several types of CRISPR systems have been coopted for use in CRISPRi (Qi et al., 2013;Specht, Xu, & Lambert, 2020;Zheng et al., 2019), but here we will focus on the commonly used Type II-A system from Streptococcus pyogenes (reviewed in Wright, Nuñez, & Doudna, 2016). In the native S. pyogenes CRISPR system, a CRISPR array containing spacers that designate target genes is transcribed into a pre-crRNA (CRISPR RNA), which is subsequently processed into individual crRNAs containing only one spacer. Processed crRNAs form a complex with a tracr-RNA (trans-activating CRISPR RNA) and the CRISPR-associated nuclease Cas9; this complex is directed to target DNA by basepairing between the crRNA spacer and a complementary DNA sequence in the target known as a protospacer. In addition to spacerprotospacer complementarity, Cas9 requires a short protospacer-adjacent motif (PAM; NGG for S. pyogenes) for DNA binding that prevents self-targeting of CRISPR arrays that lack this motif. The Cas9-tracr/crRNA complex binds to the PAM sequence, unzips the DNA duplex, anneals the crRNA and protospacer DNA, and then-if the spacer and protospacer match sufficiently-cleaves both strands of the DNA (Sternberg, Redding, Jinek, Greene, & Doudna, 2014). Doudna, Carpentier, and colleagues first showed that Cas9 is an RNAguided endonuclease and simplified the natural dual RNA system by engineering a fused tracr/crRNA known as a single guide RNA (sgRNA; Jinek et al., 2012). Cas9 can thus be programmed to target new genes of interest simply by changing the 20-nt sgRNA spacer to match a protospacer with an adjacent PAM in the target DNA.
To repurpose CRISPR as a geneknockdown technology, Qi and colleagues mutated the two nuclease active sites in Cas9, producing an inactive variant known as dCas9 ("dead Cas9"; Qi et al., 2013). dCas9 retains the ability to be directed to target genes via programmable sgRNAs but can no longer cut DNA. Instead, dCas9 inhibits transcription at the step of initiation or elongation by acting as a steric block to RNA polymerase in bacterial systems. The modest sequence requirements for CRISPRi repression-i.e., an NGG PAM sequence and adjacent spacer (Jinek et al., 2012)-and engineered Cas9 variants with altered-or relaxed-specificity PAM dependencies (Walton, Christie, Whittaker, & Kleinstiver, 2020) suggest that nearly all bacterial genes can be targeted by CRISPRi. CRISPRi systems have been established in many diverse bacteria and have primarily been used to phenotype individual essential genes in proof-of-principle work. However, CRISPRi has been increasingly valuable in targeting larger, defined sets of genes (e.g., essential genes; Liu et al., 2017;Peters et al., 2016) and in pooled phenotyping approaches at the genome scale for both model Wang et al., 2018) and nonmodel bacteria (Lee et al., 2019;Yao et al., 2020;reviewed in Vigouroux & Bikard, 2020).

Optimizing Mobile-CRISPRi transfer and integration
Distinguishing between Mobile-CRISPRi transfer and integration problems presents a challenge because both processes are required to obtain transconjugants. The efficiency of Mobile-CRISPRi transfer and integration varies by strain, such that two strains of the same species can produce vastly different numbers of transconjugants (e.g., P. aeruginosa PAO1 and PA14 differ by >100-fold; Peters et al., 2019). Cell surface features