Using CRISPRi in Escherichia coli to emphasize experimental controls in a molecular microbiology laboratory

ABSTRACT

A CRISPR interference (CRISPRi) exercise was developed for an upper-level molecular microbiology laboratory to reinforce student skills in experimental design and controls. This CRISPRi knockdown method is a variation of the commonly used Streptococcus pyogenes Cas9 system and therefore relies on similar design and techniques. Students choose and design a CRISPRi target in Escherichia coli, clone the necessary tools, and test their system with spot plating and microscopy. The motivation for introducing this unit in a laboratory course was to help close gaps in students’ broader understanding of DNA and RNA structure, primer design, bacterial gene expression, and regulation. Once introduced, this exercise became a way to help students identify, design, and rationalize proper experimental controls.

INTRODUCTION

CRISPR interference (CRISPRi) is a method for gene repression that utilizes the CRISPR-Cas9 system (1). CRISPRi tools rely on a deactivated endonuclease variant (dCas9) unable to cut double-stranded DNA (2). Like wild-type Cas9, dCas9 requires a single guide RNA (sgRNA) and protospacer adjacent motif (PAM) to bind DNA targets complementary to the sgRNA (Fig. 1). An optimized CRISPRi system for use in E. coli is easily expressed from pFD152 using anhydrotetracycline (aTc) as an inducer of dCas9 (3).

Fig 1.

Designing sgRNA for targeted CRISPRi. The 5′ region of the sgRNA is designed to bind 20 bases of the non-template of the targeted gene or operon (blue). The PAM site (5′ NGG) is identified on the opposite strand. The Shine-Dalgarno (SD) or promoter could also be targeted. Created in BioRender. Bullwinkle, T. (2025).https://biorender.com/k2tney9

Gene editing in microbial systems using CRISPR often requires donor DNAs, additional plasmid cloning, or supplemental recombineering systems (4–6). CRISPR-based protocols designed for undergraduate courses often use pre-designed sgRNAs and established reporter genes (4, 5, 7–9). The CRISPRi activity presented here provides students with autonomy and experience in critically designing their own gene targets and sgRNAs while also devising necessary experimental controls, PCR and cloning parameters, and laboratory calculations.

The upper-level course where this activity is used enrolls a total of 30–40 microbiology majors in two laboratory sections. The CRISPRi experiment is performed in six to eight laboratory sessions (2–3 h each), directly following a transposon mutagenesis module to exemplify classical and reverse genetic methods. Students have already learned the limitations of transposon mutagenesis, including how mutants with insertions in essential genes cannot be isolated and instead knockdown or regulated expression techniques can elucidate roles of essential genes. Here, students target and repress essential E. coli genes with CRISPRi. However, this exercise could be applied to any gene.

PROCEDURE

Safety issues

BSL-1 requirements for personal protective equipment, space, stock cultures, standard laboratory practices, student training, and documentation should be followed.

Lab 1: sgRNA design (1 h + shipping time)

Students are first given an independent assignment prior to laboratory work guiding them to identify an essential gene in Escherichia coli and do literature research to understand the function of the gene’s product. In class, students work in pairs to discuss each of their chosen genes and find their genomic context using the NCBI (10) and Ecocyc.org (11). Each student pair agrees on one gene as their CRISPRi target. Students consider a target sequence based on operon arrangement, gene direction, promoter(s), and a potential PAM site (Fig. 1).

Students design a sgRNA, specifically the 5′ region that will bind the genomic target. Identifying the gene’s promoter, Shine-Dalgarno site, start codon, or non-template strand reinforces student understanding of these elements. Students use an E. coli database to investigate if these elements are known and where they are (11). Knockdown is more pronounced with targets in the promoter or non-template strand, if in the coding region (2), so students are encouraged to find these regions. Students then design site-directed mutagenesis of pFD152 (gift from David Bikard, Addgene # 125546) to generate a plasmid encoding their sgRNA gene. A single PCR primer is designed by each student pair and used in combination with a common reverse primer that anneals to the sgRNA’s promoter (Fig. 2). Oligonucleotide DNA primers are ordered with 5′P (Sigma-Aldrich).

Fig 2.

PCR and cloning methodology. Primers are used to amplify most of the plasmid sequence from the sgRNA scaffold to the sgRNA’s promoter (PpflB). The 5′ end of the forward primer contains 20 bases complementary to the DNA strand to be targeted by the sgRNA. This primer sequence becomes the non-template strand for sgRNA transcription. The reverse primer is synthesized with a 5′ phosphate to allow for intramolecular ligation. pFD152 also contains a spectinomycin resistance gene for selection (not shown here) Created in BioRender. Bullwinkle, T. (2025).

Lab 2: PCR Set-up (1 h + 3 h cycling parameters)

Upon receiving their lyophilized primer, student pairs record yield and calculate the volume needed for a 100 µM solution. The tube is briefly spun before adding nuclease-free water. The primer is rehydrated for 5 min at room temperature and vortexed. For PCR, students are provided with stock and reaction concentrations (Appendix 1). Students calculate reagent volumes, working dilutions, and preparation of a master mix. Calculations are checked by an instructor prior to setup of a hot-start PCR (Appendix 1). From here on, students work independently on experimental setup to demonstrate their technical proficiency. Students prepare two reactions, one with their forward primer and one with a primer targeting rpsL, a positive control for the PCR reaction and the CRISPRi knockdown (3). Both reactions have the same reverse primer. A third, negative PCR control (no template) can also be prepared.

Lab 3: Product analysis and ligation (1-2 h + overnight incubation)

A sample of each completed PCR (5 µL) is analyzed using 0.8% agarose gels. Successful products are 9,132 bp, the same as the template, and optimally the only product size observed. Students move forward with their experimental PCR DNA, if successful. DpnI is used to digest and remove original pFD152 template. DpnI (New England Biolabs #R0176, 20 units) is added directly to the remaining PCR DNA and reactions are incubated for 1 hour at 37°C.

Intramolecular ligation is used to generate a new plasmid construct from the PCR DNA. Ligation reactions are done under dilute conditions to promote circularization and are set up using 1-2 μL of DpnI-treated PCR product DNA. Water, T4 ligation buffer (1 x), and T4 DNA ligase (New England Biolabs #M0202, 400 units) are added to a total reaction volume of 10 µL. A negative control reaction is set up with the same DNA, but lacking T4 ligase. Ligation reactions are incubated 4 h to overnight at room temperature.

Lab 4: Transformation of E. coli (2 h + overnight growth)

Chemically competent E. coli DH5α cells (Thermo Fisher Scientific #18265017) are used for heat-shock transformation and CRISPRi. For each transformation and control, 50 µL of cells (thawed on ice) are mixed with 2 µL of ligated DNA. A spectinomycin (spec) resistance plasmid (20 ng) is used for a positive transformation control and no DNA is a negative control. Cells and DNA are incubated on ice for 20 min, transferred to 42°C for 90 s, and then briefly placed on ice. SOC broth (1 mL) is added to the heat-shocked cells and incubated for 1 h at 37°C shaking. After outgrowth, cells are concentrated by centrifugation (1 min at 7,000×g), suspended in 100 µL of SOC, and plated on LB agar with spectinomycin (50 µg/mL). Plates are incubated 24–48 h at 37°C.

Lab 5: Sequencing sgRNA plasmid (1 h + sequencing time)

Individual clones from the experimental transformation plates are used to inoculate 5 mL LB with spectinomycin (50 µg/mL) and grown shaking overnight at 37°C. Plasmids are isolated using the Qiagen Miniprep plasmid isolation kit (Qiagen #12123). Concentrations of plasmid DNA are measured (Nanodrop 1000), and samples are sent for Sanger sequencing with the pFD152_Seq primer: 5′gcaattagcatattactgttacac. Results are aligned to pFD152 and sgRNA sequences using NCBI BLAST to determine the success of cloning (12).

Lab 6: CRISPRi analysis (1 h + overnight growth)

Overnight broth cultures of experimental and pFD152 control strains are used for testing CRISPRi. Serial dilutions are prepared from each culture and 10 µL of dilutions are spotted on two LB plates with spec (50 µg/mL) that contain or lack aTc. Solutions of aTc are made fresh or stored in frozen aliquots and used at 1 µg/mL. Plates are incubated for 24–28 h at 37°C. Successful completion of the CRISPRi experiment will show growth only on plates lacking aTC (Fig. 3). Lab 7 is useful for data analyses and class discussion.

Fig 3.

Loss of E. coli growth after CRISPRi. Overnight cultures of CRISPRi strains targeting essential genes gyrA or ftsZ are serially diluted and spot plated on LB plates containing spec and aTc (right) or without aTc (left). Spots are inoculated with 10 µL of each culture dilution using a paper template for guidance. The control refers to the pFD152, negative control strain.

More recently, this experiment has been extended to observe additional phenotypes. Using phase-contrast microscopy, students can check their transformants grown with and without aTc for differences in cell morphology or lysis. For example, CRISPRi knockdown of genes essential for DNA replication or cell division results in altered cell morphologies (13).

CONCLUSION

The PCR and cloning steps are successful for most students and groups work synchronously during each laboratory session. Depending on timing, students may be able to troubleshoot and repeat steps if unsuccessful on the first attempt. The positive PCR control or some of their partner’s PCR products or clones can also be used, if needed. The success of CRISPRi depends on the design of the sgRNA and sequence they targeted. Students also have the rpsL control, which provides an additional backup if students are unsuccessful with their PCR or target.

To informally assess students’ grasp of the full experiment and each of the controls, paper-based modeling is used during the first laboratory session (Appendix 4). The activity emphasizes the different DNA, RNA, and enzyme components and if they are functioning in vitro or in vivo. Guidance can then be given to groups individually, based on need, before the experiment is set up. Additional assessments evaluate student understanding of the experiment and its controls after data is collected. A brief write-up of their results allows students to communicate what their data does and does not show based on the outcomes of experimental and control samples at each step (Appendix 2). This assignment is often completed in class to promote group work and class discussion. Students will then see similar simulated PCR, cloning, and CRISPRi data on a short answer exam question, assessing individual understanding of the experiment, its controls, and data interpretation (Appendix 3).

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jmbe.00135-25.

Appendix 1 to 4. jmbe.00135-25-s0001.pdf.

PCR set-up, controls that can be introduced, sample assessment questions, and CRISPRi activity handout.

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