Part:BBa_K2839016

CRISPRi stabilized J23104 promoter

1.Short Description

This part consists of a short chimeric RNA (sgRNA), a CRISPRi stabilized promoter (J23104) and a fluorescent marker (sfGFP). The sgRNA forms a complex with dCas9, a catalytically inactive form of Cas9 and binds to the target promoter region blocking bacterial transcription initiation. This part was designed by iGEM Thessaloniki, it is RFC10 compatible and together with the dCas9 cassette can be used to achieve stabilized expression of the sfGFP marker, decoupled from gene/plasmid copy number.

2.Biology and Functionality

CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) systems are naturally found in a plethora of prokaryotic species providing resistance to foreign DNA elements. One of the widely used CRISPR systems is the one derived from Streptococcus pyogenes and is based on the cleavage activity of Cas9 which together with two RNA molecules(crRNA and tracrRNA [1] binds and cleaves a specific target site (endonuclease). dCas9 is a catalytically inactive Cas9 nuclease modified with two single point mutations which can still tightly bind at the target DNA but lacks endonuclease activity. CRISPR interference is a DNA editing technique that allows for control in gene expression. It requires the presence of a short chimeric RNA, which forms a complex with the dCas9 protein and determines the binding specificity of the complex at the target DNA.

3.Usage in our project

As a chassis for hosting the system’s devices we use DH5a E.coli cells.

Here, we utilize the ability of dcas9/sgRNA complex to tighly bind at a specific bacterial promoter sequence and act as repressor by blocking the recruitment of RNA polymerase in order to create a type I incoherent Feedforward Loop (iFFL) network motif that renders the expression of the downstream sequences independent of the plasmid’s copy number. A promoter under this effect is called a Stabilized Promoter.

Copy number positively affects GOI expression, while its repressor, being under the same positive influence, compensates for this change. If the hill coefficient that characterizes the repression is 1, and thus the repression is perfectly non-cooperative, it results in the disassociation of the GOI’s expression from the plasmid’s copy number.

4.RFC[10] Compatibility, Illegal Sites Removal

This part is RFC[10] compatible since the sequence was optimized to eliminate illegal restriction sites.

5.Design

Artificial biological systems often require multiple gene repression/multiple promoter control/multiple gene expression control (e.g. in metabolic pathways, where stoichiometry ratios are often required for the system’s proper function. Stabilizing multiple promoters using the TAL Effectors could prove difficult, as in order to achieve the desired strength-error tradeoff and the required expression level, more than one TAL Effectors would be needed. This might prove taxing to the cell, as growth reduction could occur, due to excessive metabolic burden. In our effort to overcome these issues and enrich the arsenal of tools available for stabilized promoters design we introduced the CRISPRi system.

Choosing where each component should be expressed from, we set up a model to simulate the system. Initially, we had 3 options for the design of our system, regarding dCas9 expression site/ gene placement/ gene positioning: the plasmid with the sfGFP marker, the genome, or the insertion of a second compatible plasmid. (topologies) Results showed that the expression of both the sgRNA and the dCas9 from a single plasmid (Topology A), in which the promoter to be stabilized resides, broke the system and exhibited behaviour similar to repression with a Hill coefficient of 2.

Another option was the genome insertion of dCas9 (Topology B), which would offer increased system stability, but, when simulated in our model, expression of dCas9 proved inadequate.

We decided to use a double plasmid system (Topology C), co-expressing the sgRNA along with sfGFP and expressing dCas9 on a separate vector. The plasmid containing the dCas9 expression cassette is a low-medium copy number vector (p15A ORI) and dCas9 expression rate is controlled by a pTet promoter, therefore its output depends on the usage of an external inducer, Doxycycline.

In this system, the sfGFP promoter’s stabilization was designed to depend only on the expression level of the sgRNA. Therefore, we expressed dCas9 on the highest level that creates saturating condition without inhibiting growth due to excessive metabolic burden. (citation voigt) In order to investigate the effect of dCas9 production on the growth rate of the DH5alpha E.coli strain, we conducted a growth assay, expressing dCas9 on different levels, via induction with Dox. More information can be found in the Results section.

Regarding the sgRNA design, in order to achieve the required repression level, we had 2 options: Either constitutively express a sgRNA partially complementary to the target sequence or induce the expression of a fully complementary sgRNA to its target. We decided to induce the expression of sgRNA so that we could create a response function from which we could determine the desired expression level control sgRNA expression through a L-Rhamnose inducible promoter, pRha. Full complementarity of sgRNA to its target sequence, in combination with dCas9 saturation, made it possible to determine repression levels by adding L-Rhamnose.

In order to characterize the CRISPRi stabilized promoter we induced the expression of the sgRNA driven by the L-Rhamnose inducible promoter at the desired lowest copy number (psc101).From the response function we can determine the cooperativity of the repression and -choose the expression level of the sgRNA that corresponds to the desired strength of the stabilized promoter.

After characterizing the response function of the input and output promoters, we would have settled on a desired Strength-Error level and replace the rhamnose inducible promoter with a constitutive one, thereby maintaining the expression stable, independent of the plasmids copy number, for all copy numbers without need for addition of more rhamnose to compensate for the increase in the number of RhaS binding sites. Unfortunately, we observed minimal induction when measuring the pRha mediated sgRNA expression so we hypothesized that this was due to the change of a conserved region after the promoter’s +1 site.

Therefore, we chose BBa….. for the expression of the sgRNA cassette. We opted for a strong constitutive promoter in order to achieve a low Error value as this may prove valuable when implementing the CRISPRi stabilization system in order to stabilize complex biosynthetic pathways.

5.Cloning Strategy:

After testing the response function of the CRISPRi system we replaced the rhamnose inducible promoter that is located upstream of the sgRNA cassette with the constitutive Psp1w1 promoter.In order to successfully clone the CRISPRi stabilized promoter into different plasmid backbones we followed this procedure:

• Digestion with biobrick enzymes of the Psp1w1 promoter characterization device and ligation with digested pTHSSe_59 vector. Since the Psp1w1 promoter characterization device is compatible with Biobrick RFC[10], it can be easily assembled with other RFC[10] compatible vectors. This requires digestion of the characterization device and the receiver vector with two enzymes having their restriction sites on the prefix and suffix, followed by ligation. These reactions forms the final plasmid psp1w1-pTHSSe_59 containing the Psp1w1 promoter characterization device.
• PCR amplification of the psp1w1-pTHSSe_59 plasmid with IG1, IA2 primers.These primers amplify the psp1w1 promoter sequence together with the pTHSSe_59 plasmid backbone and incorporate BsaI restriction sites flanking the amplified sequence.
• PCR amplification of the sgRNA-sfGFP-1c3 plasmid with GG1, GG2 primers. These primers amplify the sgrna cassette along with the stabilized J23104 promoter cassette and incorporate BsaI restriction sites at the external regions of the amplified sequence.
• Golden gate assembly of the amplified products.
• After digestion with BsaI restriction enzyme,the amplified sequences acquire complementary sticky ends and thus they can be assembled together forming the Psp1w1-CRISPRi-stabilized promoter-pTHSSe_59 vector.

7.Results, Characterization

• Growth assay:

In order to investigate the effect of dCas9 production on the growth rate of the DH5alpha E.coli strain, we conducted a growth assay, expressing dCas9 on different levels, via induction with Doxycycline.Specifically we inserted dcas9 expression cassette into psb3k3 vector. (p15A ORI).The dCas9 expression rate is controlled by a pTet promoter, therefore its output depends on the usage of the external inducer, doxycycline.

All measurements regarding Abs600 were performed using plate reader.Specifically, one single colony of DH5a E.coli cells containing the dcas9 expression device was inoculated into 1 ml LB+Kanamycin and grown at 37 °C, 250 rpm overnight in a MultitronPro shaker incubator.The overnight cultures were diluted 1:360 into 1ml LB+ kanamycin in 2‐ml eppendorf tubes and doxycycline was added to the following final concentration:0 ,0.2 ,0.4 ,0.6 ,0.8 ,1.2 ,4,8.The cultures were grown at 37 °C, 250 rpm in a MultitronPro shaker incubator.After 6 hours measurements for Abs600 were performed using plate reader.

Figure 1.Cell growth inhibition caused by different expression levels of dcas9, induced by different concentrations of doxycycline.

From the figure 1, it is clear that cells viability starts to significantly decline after adding doxycycline to a final concentration of 1.2 ng/ml.

• Different Copy Number Measurements:

Fluorescence intensity measurements of this construct were conducted in DH5α cells to determine the functionality of promoter stabilization over different copy numbers.

The construct was inserted in plasmid backbones with different ORIs (psc101, pUC19-derived pMB1) and transformed into DH5a cells. After that, colony PCR was performed in order to identify the colonies with the correct insert. Single colonies were picked and prepared to be measured using flow cytometry method.

• Sample preparation:

In order to prepare the cultures for flow cytometry analysis we followed the protocol created by Adam Mayer et al [1]. In particular the correct colonies were inoculated into 1 ml Lb + antibiotics and grown overnight at 37 °C in a shaking incubator adjusted to 250 rpm.The overnight growths were diluted 1:200 into 1 ml LB + antibiotics and grown at 37 °C into shaking incubator .After 2 hours the growths were diluted 1:500 into prewarmed LB + antibiotics + inducer where necessary and grown at 37 °C, 250 rpm for 5 hours.After growth, 20 μl of culture sample was diluted into 180 μl PBS + 200 μg/ml kanamycin to inhibit translation. The samples were stored at 4°C for 1 hour and then measurements were performed using the CyFlow Cube8 Sysmex Partec Flow Cytometer.

• Flow Cytometry Results:

Figure 2. CRISPRi stabilized promoter and non stabilized constitutive pT7A1w1 promoter flow cytometry fluorescence measurements at two different copy numbers. Error bars represent standard deviation from three biological replicates.

From figure 5 it is clear that sfGFP expression level, under the control of CRISPRi stabilized J23104 promoter (BBa_K2839016) with dcas9 levels induced by 0.8 ng/ml doxycycline,remains stable when expressed from vectors with different copy number Whereas,without dcas9 expression, sfGFP expression level changes when different copy number plasmids are used for its expression.

8.References:

[1]Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173–1183. https://doi.org/10.1016/j.cell.2013.02.022

[2]Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C., & Doudna, J. A. (2014). DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature, 507(7490), 62–67. https://doi.org/10.1038/nature13011

[3]Larson, M. H., Gilbert, L. A., Wang, X., Lim, W. A., Weissman, J. S., & Qi, L. S. (2013). CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nature Protocols, 8(11), 2180–2196. https://doi.org/10.1038/nprot.2013.132

[4]Bikard, D., Jiang, W., Samai, P., Hochschild, A., Zhang, F., & Marraffini, L. A. (2013). Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Research, 41(15), 7429–7437. https://doi.org/10.1093/nar/gkt520

[5]Vigouroux, A., Oldewurtel, E., Cui, L., Bikard, D., & van Teeffelen, S. (2018). Tuning dCas9’s ability to block transcription enables robust, noiseless knockdown of bacterial genes. Molecular Systems Biology, 14(3), e7899. https://doi.org/10.15252/MSB.20177899

[6]Nielsen, A. A., & Voigt, C. A. (2014). Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks. Molecular Systems Biology, 10(11), 763–763. https://doi.org/10.15252/msb.20145735

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 194
  Illegal NheI site found at 463
  Illegal NheI site found at 486
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1042
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]