Team:Groningen/Design
Design
Introduction
The model results allow us to assume with reasonable certainty which spacers are likely to get incorporated. But more broadly, what is actually required for a CRISPR based bacteriophage detection system?
First off, the bacteriophage sequence has to get incorporated into a spacer array. This requires several different Cas proteins. We worried that the natural spacer adaptation rate would be too low, so we opted for an improved, hyperactive, hCas9 protein together with Cas1, Cas2 and csn2. The altered version displays improved features, especially concerning the speed and therefore the quantity of spacer incorporation. We expect the likelihood of the correct spacer to be adapted to increase proportionally with an increase in total quantity of adaptation.
This subsystem would already allow for resistance and in essence mimics the natural CRISPR immune system. But then again, we do not wish to replicate the natural CRISPR mechanism but utilize its potential for a detection mechanism.
This leads to our other subprojects, the dCas9 and the reporter system. The gRNAs that the hCas9 array produces can be used by a catalytically dead Cas9 protein as well. The gRNAs allow the dCas9 to target the reporter plasmid. This plasmid contains spacer inserts upstream of a GFP protein. Binding of the dCas9 protein will sterically hinder transcription of the GFP gene causing a detectable decrease in fluorescence. Essentially, if the matching protospacer gets incorporated into the array and then leads to a decrease in GFP fluorescence, you can safely assume the presence of the respective bacteriophage.
In the following paragraphs, we would like to elaborate on the manner how we envision our design and system to look like in more detail and how much we achieved during our project.
hCas9
- The first gblock was used to remove the XbaI site and introduce the iGEM prefix and suffix. This part was first placed in the pSB1A3 vector to form the backbone for the Gibson assembly. The plasmid had to be linearized using PCR before it could be put together with the other fragments.
- The second gblock was used to introduce the I473F mutation and remove the EcorI site.
- The PCR fragments that were amplified from the pWJ40 plasmid were designed in such a way that they already contained the overhangs for the Gibson assembly.
Biobrick construction
As a basis for the biobrick compatible hCas9 operon we used the Cas9 CRISPR operon from Streptococcus pyogenes. To make this into a biobrick compatible hCas9 operon we had to introduce the I473F mutation [(Heler (2017)], remove the prohibited restriction sites it contained and attach the biobrick prefix and suffix.To achieve this we used a combination of synthetic DNA (gBlocks) and DNA fragments that were PCR amplified from pWJ40 (containing the Cas9 operon, plasmid map), which both our supervisor Chenxi gave us. All fragments were finally put together using Gibson assembly. All cloning steps were performed in E. coli DH5α. Figure 1 gives an overview of all the cloning steps that were used.
To combine all the fragments that we created we used a Gibson assembly. After transformation the product into E. coli DH5α and isolation of the plasmid, several PCR reactions were performed with different primers to confirm that it contained the desired product. After the gels seemed to be correct we sent the plasmid for sequencing and further verification. Unfortunately, it turned out that the amplified I473F gblock part of the gene contained the wrong sequence. Due to time constraints of the parts submission deadline, we were not able to construct a new biobrick compatible hCas9 operon. We encourage next iGEM teams to pick up where we left as hCas9 seems to be a valuable addition to the iGEM registry!
Besides the hCas9 operon the tracer RNA and the spacer array are also required to get a successful CRISPR response. In this project we constructed biobrick compatible tracer RNA and several pre-programmed spacers arrays. We ordered the tracer RNA as a gblock in combination with the constitutive lactococcal promoter pUSP45. The sequence of the tracer RNA gene was taken from the S. Pyogenes genome and the sequence of the pUSP45 promoter was provided by one of our supervisors. Since the tracer RNA is functional as RNA, and has no start codon, we had to precisely position the tracer RNA gene relative to the promoter (tracer RNA).
Validation construction
As mentioned earlier we were not able to construct the biobrick compatible hCas9 operon. So the experiment that will be described next was not executed. To validate that hCas9 is capable of acquiring spacers in both Lactococcus lactis and E.coli, we wanted to perform an on-plate acquisition assay. In this assay we would have measured the rate at which spacers would be acquired. For this assay we first would have inserted the hCas9 operon into an inducible expression vector. We were planning to use an arabinose inducible pBad vector for E. coli and a nisin inducible pNZ8048 vector for L. lactis. For comparison we would also have included the regular Cas9 operon. The cells would then be exposed to bacteriophages and the surviving colonies isolated. Using PCR we could then have measured the increase in size of the spacer array. The amount of surviving colonies and the sizes of the spacer arrays would have given an indication about the efficiency/activity of the hCas9 operon in L. lactis and E.coli.dCas9
- To start off the dCas9 was PCR amplified out of the plasmid using primers to incorporate XbaI as well as a PstI restriction sites in front of and behind dCas9, enabling dCas9 integration into an iGEM Vector.
- Next we removed the EcoRI site, since this was still present in the Addgene plasmid and interfering with Biobrick compatibility. In order to accomplish this, two sets of quick-change primers were designed (image of sequence dCas9 plasmid part containing EcoRI site and all four primers). This created the regular dCas9, which can be seen as an improvement on (BBa_K1026000).
- Since all mutations are positioned at the end of the gene, a gBlock was designed containing the end of dCas9 with all four mutations. To exchange the gblock with the original end of dCas9 the BamHI restriction site was used. The end of the gblock contained the biobrick suffix. To ensure that only mutated dCas9 would be transformed, the correct fragment was subjected to a gel-extraction after restriction of the pSB1C3 dCas9 plasmid. From this sub-project both the biobrick-compatible dCas9 (BBa_K23610000) and dCas9-VRER (BBa_K2361001) were submitted to the iGEM HQ.
Biobrick construction
For the next part for our project we created a biobrick compatible dCas9 protein that would recognize different PAM sites than hCas9. To achieve this we had to introduce the VRER mutations into the dCas9 gene, that were described by [Kleinstiver (2015)].
We started out by trying to transform the dCas9 from the biobrick dCas9-Ω (BBa_K1723000), which was provided in the iGEM distribution plate, as a basis. However, we were not able to recover successful transformants. Therefore we decided to pursue another strategy utilizing the addgene plasmid pJWV102-PL-dCas9 (plasmid map), which was supplied to us by our supervisor Chenxi.
Validation
To validate that dCas9 worked we designed an experiment in which we would be able to determine if dCas9 was capable of interfering with the expression of GFP. Since the spacer array and the tracerRNA are not on the same plasmid we used pre-designed guide RNA’s. The guide RNA’s were designed so they would bind in the coding sequence of GFP after a constitutive promoter WHICH promoter IS THIS MART???. We did not only want to see if our dCas9 could interfere with gene expression, we also wanted to see if the VRER mutations would cause it to recognize a different PAM sequence. So half of the guideRNA’s had a PAM recognition sequence for dCas9 and the other half had the recognition sequence for dCas9VRER. This way we could compare the relative amount of GFP inhibition between the guide RNA’s and determine of the VRER mutations changed the recognition sequence for dCas9.Reporter
Biobrick construction
The process of creating the dCas9 and tracRNA parts is described elsewhere and they could be used without further modification. The target array for the reporter plasmid and crRNA array were custom designed and synthesized for use in this sub-project.The cRNA array design was based on the natural S. Pyogenes CRISPR locus. The array was reduced down to a single spacer and the natural promoter was exchanged for the lactococcal pUsp45 promoter. Outward facing BsaI were inserted to allow the crRNA array to be easily reprogrammable by insertion of a short oligo. Furthermore, a termination sequence proven to work in L. lactis was placed after the putative S. pyogenes terminator and the whole was flanked by biobrick adapters. The whole was ordered for synthesis from IDT, see here. We submitted the completed crArrays (BBa_K2361004), empty (BBa_K2361007) with spacer 21 (BBa_K2361006) and spacer 20 (BBa_K2361005) .
The reporter was designed to use a fluorescent protein (sfGFP) that has been shown to show robust expression in L. lactis. The sfGFP transcript is driven by the lactococcal p32 promoter and followed by two termination sequences. Because of the limited synthesis size available to us the reporter target array and sfGFP gene “unit” could not be ordered in its completed form. We shrunk our sequence by removing a chunk of the sfGFP sequence. We designed it such a manner that this created a new HindIII restriction site that would be lost upon insertion of the missing sequence. The missing sequence was amplified from an entry from the 2014 Groningen iGEM team (BBa_K1365020). As with the crRNA array, the sfGFP reporter was flanked by a biobrick prefix and suffix. We were able to insert the first sfGFP from the gblock into the PSB1C3 backbone
Between the transcription start site of p32, and the RBS and start codon of sfGFP two Cas9 target sequences had to be placed. These 20nt target sites, “20” and “21”, were predicted by our bioinformatics approach to be good contenders for uptake by the CRISPR operon and were created separately. They were flanked by a -NGCG- PAM to allow them to be recognized by the PAM targeting mutant dCas9VRER. Like the crRNA array, outward facing BsaI sites were added to allow the target sites to be exchanged with a short oligo. Unlike the BsaI sites in the crRNA these restriction sites in the reporter target array did not interfere with its intended function. Therefore, they could be left in for the initial test and called upon later if the target array needed to be exchanged. In the end we were able to construct the different CRISPR arrays with a pUsp45 promotor and 20, 21 or empty spacers.
Validation
To experimentally validate this part, several of our other subprojects have to be completed as well. The spacer acquisition subproject has to incorporate the appropriate spacer (20/21), transcripe the crRNA together with the tracRNA which should allow the dCas9VRER to target the reporter plasmid which leads to a measurable decrease in GFP fluorescence. This experiment can be performed in a standard 96-well platereader which is able to measure GFP fluorescence, maintain the required temperature as well as measure the optical density at 600nm. Due to time concerns we were not able to perform this feat experimentally.
Lactis toolbox
Biobrick construction
As our detection system is designed to ultimately by integrated into L. lactis, we wanted to provide the registry with the desired promoters which were not available in a pSB1C3 backbone. For each promoter, a different plasmid and primer pair was used to amplify the sequences from their native backbones. The pNisA lactococcal promoter was amplified from the pNZ8048 plasmid using the G65 and G66 primers [2]. The p32 lactococcal promoter was amplified from the pMG36E plasmid using the G67 and G68 primers[7]. The pUsp45 lactococcal promoter was amplified from the already cloned part BBa_K2361003 using the G63 and G64 primers. This added the biobrick restriction sites combinations to the flanks of the promoter sequence. This allowed us to incorporate the promotor sequences into the biobrick-compatible format. In the end the sequencing data for p32 & pUsp45 did not match the expected sequence, so we resorted to only submitting (BBa_K2361009) and testing PnisA activity.Validation
So how do we actually validate our promotor? Since iGEM requires biobrick submissions to be contained in the PSB1C3 vector, the first step was converting this shipment format to a genetic setup which is suitable for protein expression. Since the sequencing data for p32 & push45 did not match the expected sequence we resorted to only testing PnisA activity. As the lactococcal expression vector already contains the pNisA promoter, this was used as the backbone for our measurement setup. The most convienent way to characerize the promoter was to let it express a fluorescent protein. Therefore, GFP was cloned behind this promoter within the pNZ8048 vector. The strenght of the promoter was tested by inducing the L. lactis cells with various concentrations of nisin, ranging from 1 ng/μl to 80 ng/μl. This gave us valuable insight into the capabilities of this inducable promoter. See the result page for the results.References
- Heler, R. et al. Mutations in Cas9 Enhance the Rate of Acquisition of Viral Spacer Sequences during the CRISPR-Cas Immune Response. Mol. Cell 65, 168–175 (2017).
- Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015)
- Cloning overviews were made with SpapGene software.
