20.109(F19):Induce CRISPRi system (Day7)

Introduction

The CRISPRi system involves three genetic elements: the target gene within the host genome, the pdCas9 plasmid, and the pgRNA_target plasmid. Though the target genome is native to the host cell, the plasmids must be transformed into the cell and maintained using antibiotic selection. Throughout this module, we have learned about and worked with the CRISPRi plasmids as individual units, but now we will consider the system as a whole in the context of gene regulation.

In the previous laboratory session, you co-transformed pdCas9 and your pgRNA_target plasmids into E. coli MG1655. The colonies present on your LB plates containing chloramphenicol and ampicillin should carry both plasmids in that they were able to survive selection by both antibiotics. The promoter (pJ23119) driving expression of your gRNA sequence in the gRNA_target plasmid is constitutively active. This means that RNAP is not prohibiting from binding and transcription of the gRNA target sequence is therefore not inhibited. Therefore, your gRNA_target is always present in the MG1655 cells and binding to the target sequence within the genome.

Schematic of aTc induction of tet promoter.

In contrast, expression of the gene encoding Cas9 within pdCas9 is regulated by an inducible promoter (p_LtetO-1). An inducible promoter is 'off' unless the appropriate molecule is present to relieve repression. In the case of our system, expression of the gene encoding Cas9 is inhibited due to the use of a tet-based promoter construct. Tet is shorthand for tetracycline, which is an antibiotic that inhibits protein synthesis through preventing the association between charged aminoacyl-tRNA molecules and the A site of ribosomes. Bacterial cells that carry the tet resistance cassette are able to survive exposure to tetracycline by expressing genes that encode an efflux pump that 'flushes' the antibiotic from the bacterial cell. To conserve energy, the tet system is only expressed in the presence of tetracyline. In the absence of tetracycline, a transcription repressor protein (TetR) is bound to the promoter upstream of the tet resistance cassette genes. When tetracycline is present, the molecule binds to TetR causing a confirmational resulting in TetR 'falling off' of the promoter. In the CRISPRi system, the tet-based promoter construct upstream of the gene that encodes Cas9 is 'off' unless anhydrotetracyline (aTc), an analog of tetracyline, is added to the culture media.

Taken together, the gRNA-target molecule is constitutively transcribed and, as stated above, always present / binding to the target. The dCas9 protein is only present when aTc is added. Thus, gene expression is only altered when aTc is present. As represented by the schematic below, the gRNA_target 'seeks out' the target within the host genome and recruits dCas9 to the site. When associated with the target / gRNA_target complex, dCas9 binds to the site and acts as a 'roadblock' by prohibiting RNAP access to the sequence. Because the gene of interest is not able to be transcribed, the protein encoded by that gene is not synthesized. In our experiments, we hypothesize that the absence of specific proteins, or enzymes, in the fermentation pathway will increase the yield of either ethanol or acetate.

Schematic of CRISPRi system induction. Following addition of aTc, the dCas9 protein is produced and recruited by the gRNA target sequence to the target gene. Once recruiting, the dCas9 protein associates with the gRNA/genome complex and impedes transcription by RNAP.

Protocols

Part 1: Communication Lab workshop

Our communication instructors, Dr. Sean Clarke and Dr. Prerna Bhargava, will join us today for a workshop on organizing and writing your Research article.

Part 2: Examine pgRNA sequencing results

Your goal today is to analyze the sequencing data for you two potential mutant pgRNA clones - two independent colonies from your amplification reaction - and then decide which colony to proceed with for the CRISPRi manipulation of the E. coli MG1655 fermentation pathway.

Retrieve sequence results from Genewiz

1. Your sequencing data is available from Genewiz. For easier access, the information was uploaded to the Class Data Page.
2. Download the zip folder with your team sequencing results and confirm that there are 8 files saved in the folder.
3. For each sequencing reaction, you should have one .abi file and one .seq file.
4. Open one of the .abi files.
   • This file contains the chromatogram for your sequencing reaction. Scroll through the sequence and ensure that the peaks are clearly defined and evenly spaced. Low signal (or peaks) or stacked peaks can provide incorrect base assignments in the sequence.
5. Open one of the .seq files.
   • This file contains the base assignments for your sequencing reaction. The bases are assigned by the software from the chromatogram sequence.
   • The start of the a sequencing reaction result often contains several Ns, which indicates that the software was unable to assign a basepair. Given the chromatogram result, why might the software assign Ns in this region of the sequence?
6. Include all of your observations in your Benchling notebook. You can also attach the files to your entry.

Confirm gRNA sequence in pgRNA using SnapGene

You should align your sequencing data with a known sequence, in this case the gRNA target sequence you selected, to identify any unintended base changes that may have occurred. There are several web-based programs for aligning sequences and still more programs that can be purchased. The steps for using SnapGene are below. Please feel free to use any program with which you are familiar.

1. Generate a new DNA file that contains the gRNA oligo you designed on M2D3. This file should contain only the target sequence you selected and the dCas9 tag sequence (not the plasmid sequence).
2. Generate an additional new DNA file that contains the results from the sequencing reaction completed by Genewiz.
   • For each sequencing result you should generate a distinct new DNA file. Remember you should have a forward and reverse sequencing result for each of your clones!
   • Paste the sequence text from your sequencing run into the new DNA file window. If there were ambiguous areas of your sequencing results, these will be listed as "N" rather than "A" "T" "G" or "C" and it's fine to include Ns in the query.
   • The start and end of your sequencing may have several Ns. In this case it is best to omit these Ns by pasting only the 'good' sequence that is flanked by the ambiguous sequence.
3. To confirm the gRNA sequence in your clones, open one of the forward sequencing results files generated in the previous step.
   • Select 'Tools' --> 'Align to Reference DNA Sequence...' --> 'Align Full Sequences...' from the toolbar.
   • In the window, select the file that contains the gRNA oligo sequence and click 'Open'.
4. A new window will open with the alignment of the two sequences. The top line of sequence shows the results of the sequencing reaction and the bottom line shows the oligo you designed.
   • Are there any discrepancies or differences between the two sequences? Scroll through the entire alignment to check the full sequencing result and note any basepair changes.
5. Follow the above steps to examine all of your sequencing results. Remember: you used a forward and a reverse primer to interrogate both potential gRNA_target plasmids.
6. You should save a screenshot of each alignment and attach them to your Benchling notebook.

If both clones for your gRNA_target have the correct sequence, choose either co-transformant to use for the aTc induction step. If only one is correct, then this is the co-transformant you will use next time. If neither of your plasmids carry the appropriate insertion, talk to the teaching faculty.

Part 3: Prepare media for mixed-acid fermentation inoculations

To test the effect of your gRNA on altering the production of either ethanol or lactate, you will incubate the co-transformed E. coli cells both with and without oxygen. Remember from lecture that cells grown anaerobically will produce more fermentation products; however, our goal is to further enhance the production of these products by manipulating the fermentation pathway.

1. Acquire 4 glass culture tubes and 4 15 mL conical tubes from the front laboratory bench and label as shown below:
   • MG1655 +O₂ -aTc (glass tube)
   • MG1655 -O₂ -aTc (15 mL conical tube)
   • MG1655 +O₂ +aTc (glass tube)
   • MG1655 -O₂ +aTc (15 mL conical tube)
   • MG1655 +CRISPRi +O₂ -aTc (glass tube)
   • MG1655 +CRISPRi -O₂ -aTc (15 mL conical tube)
   • MG1655 +CRISPRi +O₂ +aTc (glass tube)
   • MG1655 +CRISPRi -O₂ +aTc (15 mL conical tube)
2. Be sure to include your team information on each tube!
3. Obtain a bottle of LB broth from the front laboratory bench.
4. Transfer 5 mL of the media into each tube labeled for inoculation with MG1655 (alone, not co-transformed).
5. Calculate the volume of the chloramphenicol and ampicillin antibiotic stocks that are required to maintain the pdCas9 and pgRNA plasmids, respectively, in the remaining volume of the LB media using the information below:
   • For chloramphenicol: the stock concentration is 25 mg/mL and the final or 'working' concentration should be 25 μg/mL.
   • For ampicillin: the stock concentration is 100 mg/mL and the final or 'working' concentration should be 100 μg/mL.
6. Add the appropriate volume of each antibiotic stock to the LB aliquot and mix thoroughly.
7. Transfer 5 mL of the media you prepared in Step #5 into each tube labeled for inoculation with MG1655 +CRISPRi.

On either Monday or Tuesday afternoon, depending on your laboratory section, the teaching faculty will inoculate MG1655 or a co-transformant into your culture tubes and add 2 μM aTc (final concentration) to the appropriate tubes. All tubes will then be incubated at 37 °C until you return for the next laboratory session.

Reagents list

• Luria-Bertani (LB) broth: 1% tryptone, 0.5% yeast extract, and 1% NaCl
• chloramphenicol antibiotic stock (25 mg/mL)
• ampicillin antibiotic stock (100 mg/mL)
• anhydrotetracycline (aTc) stock (1 mM) (from Sigma Aldrich)

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