Team:Moscow City/Engineering

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Engineering success

Our system aims to detect a gene affecting the spread of the dangerous EHV virus in the saliva of horses. We are creating the first RNA test for EHV. RNA detection is good because in doing so we diagnose an actively replicating virus, which communicates the danger and that the horse is capable of infecting others.

Among the possibilities, we considered EHV types 1 and 4 because they are the most pathogenic. We then looked for protein-coding sequences that would either be shared exclusively with types 1 and 4 or would be unique to each of these virus types - and what we found shared was a region associated with the late life-cycle genes of the virus: ORF3 tegument protein (genome position 2434 - 2512) Targets were matched 20 bp apart for detection with paired dcas13 (catalytically inactive cas13) and split beta-lactamase (with inteins or not). This is how we came to be able to specifically detect EHV-1 and type 4 RNA in a cell-free system (in vitro).

gRNA (dcas13a-N_lact):
gRNA (dcas13-C_lact)
We have decided to check two potentially suitable systems:
  • paired dcas13a, connected with beta-lactamase fragments
  • paired dcas13a, connected with beta-lactamase and intein fragments. Inteins allow protein splicing, which increases the probability of connecting two beta-lactamase halves in one protein. Such constructions have not ever been presented on iGEM.

Research: We investigated and found that you can assemble beta-lactamase fragments in two different ways: we called them intein and non-intein.

System design
Intein system:
When two proteins get closer due to landing on viral RNA, trans-splicing happens, and peptide bond forms between beta-lactamase halves with also forming an active enzyme. It cleaves nitrocefin and the appearance of red coloration indicates the presence of the viral RNA in the solution.
  1. Scheme of the intein construct
  2. Scheme of trans-splicing
For the system without intein:
Full beta-lactamase forms when two proteins get closer due to landing on viral RNA. It splits nitrocefin, and the appearance of the red color signals us that there is a viral RNA in our solution.

  1. Scheme of the non-intein construct
Design: When choosing between different Cas-proteins, we decided to work with LwadCas13a protein capable of detecting RNA because LwadCas13a was already available in our host lab, so we focused on our initial gRNA and protein design around it.

We chose beta-lactamase as a reporter protein because it is well-known and reliable. We also had it encoded in AmpR in our lab.

We also understood that we need to place beta-lactamase fragments far from dcas13a to not interfere in each other’s functions. Therefore, we added a medium-sized flexible linker consisting of neutral amino acids: GGGGGG.

You can read more about systems choosing here:
BBa_K4088888, BBa_K4088889, BBa_K4088890, BBa_K4088891.

During the modeling of our experiment, we have been choosing between two ways of cloning: sequential ligation and Golden Gate.

We chose Golden Gate for dCas13 and beta-lactamase fragments insertion. Golden Gate does cloning: to introduce multiple inserts - thanks to which one does not have to do screening after each insertion, which saves development time. In addition, with linear ligation, the yield is negligible due to the inefficiency of ligation and the small amounts of DNA extracted from the gel. Thus, we increase the yield of DNA using the Golden Gate.

Build: We will use Golden Gate cloning to assemble our both systems. For the intein constructs, the intein fragments will be added by restriction/ligation at sites close to the AmpR gene fragment (see the Build section for details).

In the end, we will get genes of each chimeric protein. We will express dcas13a-C_lact and dcas13a-N_lact from pet-28(+) и pet-30(+) plasmids due to the convenient location of His-tags and restriction sites. Also, the pET system is the most powerful system yet developed for the cloning and expression of recombinant proteins in E. coli. We will also express similar chimeric proteins with intein hanging fragments dcas13a-C_lact-C_int and dcas13a-N_lact-N_int. All four proteins will be expressed in an expression culture that is often used in our lab, Rosetta (suitable for protein expression with pET).
In addition, we simulate the interaction of beta-lactamase fragments and intein fragments for a preliminary comparison of the efficiency of our systems.

Test: During the experiments, we will test the efficiency of both systems by comparing the time it took solutions with different protein systems to change their staining intensity to some fixed value using a multi-well spectrophotometer. If the binding efficiency of the beta lactamase fragments without split inteins is low, we may not wait for a change in staining - and then there will be false negatives. Therefore, we will also set up a real time PCR to detect the RNA of the virus in solutions. Thanks to the data obtained, we will make a conclusion about the efficiency of the detection systems used. We will also use several controls (more details in the Test section)

Learn: In the end, we will choose the best detection system.
Our goal is a test strip for virus detection. We also want to make the test sufficiently accurate and as fast as possible - for this, we use HUDSON so that we don't have to perform RNA extraction. However, we will start with a plate with a freshly prepared chimeric protein solution with nitrocefin in the wells, to which we will add the RNA of the virus after LAMP (loop-mediated isothermal amplification). We also aim at the fact that the use of our test will not involve the use of spectrophotometry, but at the stage of development, it should be used for comparison, calibration of the systems.
See our lab book page
mOdeling page
Primers were selected using SnapGene.
We checked Melting temperatures in the NEB Tm Calculator online software.

In SnapGene, we modeled molecular cloning of four plasmid genetic constructs producing fusion protein. They included four plasmid assemblies.Two of them contained: the N- or C-terminal half of the beta-lactamase gene combined with the catalytically inactive dCas13a using a polyglycine linker. The other two had the same construct, including the N- or C-terminal half of the intein together with the N- or C-terminal half of the beta-lactamase, respectively.

Download SnapGene history

In the lab, we have started to build our system: we have now successfully cloned the dcas13a-C_lact gene. See the lab book for more details

We could not complete the wet part of the project in the laboratory due to the closed access and the lack of time. However, we assume that all experiments can be carried out when access becomes available again.

Where did we stop in the lab:
On the last day, we finally grew colonies on all the Petri dishes.
We stopped at the stage of screening colonies, which we transformed with plasmids with expected inserts after Golden Gate Cloning dCas13a-C_lact, dCas13a-N_lact, dcas13a-C_lact(int) и dcas13a-N_lact(int).

Here’s what we anticipate as the next steps:
1. Screening:
1.) PCR of the colonies up to 8 on each petri dish with Quick-Load® Taq 2x Master;
2) Mix - in this case we amplify beta-lactamase with screening primers - because otherwise, we would have to amplify an insert of about 4 kbp, and in our case - about 600bp (presence of dcas13 is confirmed indirectly).
restriction analysis;
3) Sanger - sequencing (quick and cost-effective).

2.1 Additional following actions for constructs with inteins:
1) We need to add split-inteins using restriction/ligation (N-int PsiI- blunt ends ligation) There is no need to phosphorylate because we have two incompatible restriction sites. And the vector will not close on itself. And also because we don't have PCR and due to restriction all the 5' phosphate and 3' hydroxyl are present;
2) Chemically transforming plasmids containing the insertion dcas13a-C_lact-int и dcas13a-N_lact-int в Heat-Shock competent Top10 for plasmids. (Top10 Heat Shock competent cells are already available in our lab and frozen at -80°C);
3) Cells are spread on Petri dishes on chloramphenicol (according to the protocol NEB) and incubated for 16 hours at 37°C.

2.2 Screening the Top10 colonies:
1) PCR screening, including amplification of inserts (beta-lactamase fragment and intein - total length is short;
2) Restriction analysis and we only need to check the orientation of the insertion for N-intein, because it is inserted by the blunt site;
3) Sanger-sequencing.

General next steps for:
dcas13a-C_lact_int, dcas13-N_lact-int, dcas13a-C_lact и dcas13-N_lact:

3.1 Restriction and ligation:
1) dcas13a-C_lact_int into pET-30a(+) (because the His-tags are positioned correctly there and because pETs are ideal systems for expression, as we wrote about above);
2) dcas13-N_lact-int into pET-28a(+) (same reason);
3) dcas13a-C_lact into pET-30a(+);
4) dcas13-N_lact into pET-28a(+).

3.2 Transformation of ligase mixture with DNA sequences of chimeric proteins in Rosetta cells;

3.3 Protein expression at 18 degrees in 3.5 liters of LB with the addition of IPTG.

You can read more about the plan of experiments below in Lab protocols.

In advance, in case of difficulties, we made a file with tips and solutions from NEB: Troubleshooting on Protocols.

Next, we give the flasks to the group that does the protein isolation in our lab and tell them the following information:

1) Protein isolation should be done on Ni-NTA agarose by His-tags;
2) Amino acid sequence of the product;
3) Molecular mass of the product (check amino acids sequences pdf);
4) The isolated protein will be stored in the buffer:
  • 0.3 M NaCl
  • 20 mM Tris-HCl, pH 8.0
  • 0.5 mM EDTA
  • 1 mM DTT
  • 50% Glycerol

gRNA and the target site of the viral RNA we will buy ready-made because this way there is a guarantee that we will definitely get the RNA in a short time. For the further realization of the test, we will get EHV-1 and EHV-4 samples from our European colleagues.

In addition, we need to consider the need to use the control at each step of the test, to which we will not add viral RNA. This is due to the following: if a lot of chimeric protein: then the inteins will combine earlier, even though trans-splicing is a low-efficiency process. And in vitro detection of dcas13a target RNA is a fast-going reaction. Thus, the ribonucleoprotein complex quickly finds the RNA in the right place and sits on it. The beta-lactamase fragments converge with the inteins, and trans-splicing occurs because of the spatial proximity of the intein fragments (in the case of the non-intein system, the beta-lactamase fragments converge and interact).

We will check the work of our detection system in stages:
First, we need to assemble our system from all the components we have. To do this, we first place separately in 4 test tubes in a special buffer different chimeric proteins and the gRNA corresponding to each one. After a minute, these solutions can already be used, since the dCas13a protein is joined to the gRNA in almost two minutes.

Mix in pairs the solutions of the intein system and separately the non-intein system. Then we quickly add viral RNA to the protein solutions - to all but the control. Then we add 3-5 drops of 0.5 mg/ml Nitrocefin to 1 ml of broth suspension. The appearance of red color within 20-30 min. Indicates beta-lactamase activity.

All concentrations of the components must be chosen experimentally. However, we can rely on our experience with similar systems and take preliminary values of the component concentrations from the articles:

  • 3 μL 1000nM gRNA (100nM final) and 2 μL 0.25uM fusion protein into 8.5 uL of 1X HEPES buffer (20mM HEPES, 150mM KCl, pH 7.5), and incubated at 25℃ for 10 mins.
  • Add 3μL 30nM target (3 nM final) to the mixture of two protein-gRNA complexes and incubate at 37°C for 30 minutes.
  • Pipet samples (30uL per well) into a 96-well black-welled plate (warmed to 37°C)

In our case, 37°C is the optimal temperature for dcas13a and for protein trans splicing, which is important for the intein system.

In a separate stage of the test preparation we need to know the detection threshold of the system. We will perform a series of dilution on the viral RNA before lamp-amplification and find the detection threshold value when the PCR products are subjected to the measurement: 0.01 cp/ml (copy/ml); 0.1 cp/ml; 1 cp/ml; 10cp/ml; 100 cp/ml; 1000 cp/ml; 10000 cp/ml;

Controls on the stage of development:

  1. Nitrocefin should only stain from beta-lactamase. Therefore we will test the solution with all components except our proteins - if the solution turns red, it means it is contaminated with another substance (like ampicillin) or there is something wrong with the nitrocefin and we need to replace it.
  2. The solution without viral RNA will stain, but over a long period of time due to accidental convergence of inteins in the solution - this will be a positive control (samples will be compared to it every time);
When testing a real sample of a sick horse, it is necessary to purify the RNA from the saliva because substances in the saliva would interfere with further steps. After reverse transcription and preparation and sample preparation by the HUDSON method, LAMP amplification is performed because there was not enough RNA in the saliva after all.

If our system successfully detects the presence of viral RNA, we will see a change of the color in solution in the well of the plate from yellow (OD ~380nm) to red (OD 490nm). Color intensity should be detected by a multi-well spectrophotometer.

Regarding the detection limit: if the minimum detectable amount of viral RNA will give a nitrocefin staining intensity, which will not visually easily differ from the control - it will be necessary to use colorimetric detection methods. If it will differ markedly, in which case the test remains portable, and no equipment for detection is needed - it is only needed at the development stage.

Proposed implementation steps of our test:

  1. We take a sample of RNA from the horse's nasopharynx;
  2. Extract the RNA using HUDSON method (20 min, more information below);
  3. Reverse transcription and LAMP (20 min);
  4. Perform T7 polymerase transcription (20 min);
  5. We take our paired ribonucleoproteins dCas13a/gRNA and load them on the plate with the sample and control without the virus (40 min);
  6. Recognition: Add nitrocefin. If the color of the sample changes to red, the viral RNA is present in the sample. If the color of the sample and control are the same then viral RNA isn't present (10 min).

HUDSON finished urine or saliva could be directly added to LAMP reaction mixtures with no dilution or purification step without inhibiting subsequent amplification or detection.

We chose the HUDSON method, which became widespread during the COVID-19 epidemic. The essence of the method is to heat the sample to destroy nucleases, inactivate pathogens and release RNA by thermal and chemical denaturation. P

LAMP: Due to the high rate of product formation (>10 µg, >50X PCR yield) within a short period of time (10–15 minutes), LAMP has detection sensitivity comparable to or in many cases, exceeding that of real-time quantitative PCR (qPCR)-the most common “gold standard” technique in most molecular diagnostic assays. LAMP is insensitive to contamination of the solution, and it is possible not to purify the RNA from the mixture.

Targeted viral RNA sites were selected, through binding to which our paired dCas13 system would provide specific detection of EHV-1 and EHV-4 viruses.

Molecular dynamics of the beta-lactamase cleaved into two fragments were performed with a trajectory length of 20 ns, from which the free fragment binding energy was calculated by the MM-PBSA method. The energy was negative, indicating that the formation of their complex is energetically advantageous.

We have determined the most likely mutual arrangement and their conformation relative to each other. The minimum energy shows that this is the state most 'convenient' for proteins. We can see with high probability this very complex, but its further behaviour in the buffer will only be shown by dynamics. It will also show binding energy. Binding energies and pathway analysis can already point to the biological meaning and biological effect.

We obtained one component of the system: it is dCas13-C_lact-int - it successfully passed the screening stage. But its obtaining caused us some difficulties due to the inefficiency of ligation. To overcome this, instead of the 1h elongation suggested in the protocol, we performed it for 12h and the result was positive. We will take this into account in the future.

At the same time, we developed a detailed plan for performing experiments in the laboratory to implement our idea

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