Team:Paris Bettencourt/Project/Detect

   We developed a sensor to detect whether a particular bacterial strain carries a specific antibiotic resistance gene. Our sensor system consists of E.coli lab strain and of a synthetic phagemid with a CRISPR/Cas system and LacZ as a reporter under the control of a pRecA promoter (SOS response promoter).
If our CRISPR/Cas system can bind to the target (antibiotic resistance gene), the Cas9 generates at this specific target site a double strand break, which will induce the expression of our double-strand break SOS sensor (Figure. 1). Having the system on a phagemid, the sensor system will spread all over a population, to get a clear color output if the target has been detected. Depending on target sequence the system carries, we can identify different antibiotic resistances in a strain. This is a novel approach of detecting genes in bacterial strains. We used E.coli and a M13 phagemid to target the kanamycin resistance gene.
This sensor is a proof of concept for a similar system in Mycobacterium tuberculosis. Such a system could potentially be used to test if a patient has TB and what type of resistance genes the specific strain contains to adapt the patient’s drug treatment.

   Tuberculosis (TB) remains a major global health problem. While the treatment of this disease in countries with adequate medical care is fairly easy to detect and treat, it remains hard to treat and diagnose in poorer countries.
Up to now, a high quality lab that uses modern diagnostics is a prerequisite for early, rapid and accurate detection of TB. Therefore, diagnosis of TB and drug resistant TB remains a particular challenge for laboratory systems, especially in developing countries.
The lack of cheap, quick and accurate tests make it hard to control the Tuberculosis epidemic, which claims millions of lives every year in developing countries.
Setting up a cheap, fast and culture-based method could therefore decrease diagnostic time and facilitate patient treatment.
The most common method for diagnosing TB nowadays is sputum smear microscopy, in which bacteria are observed in sputum samples of patients under the microscope. However, this cannot be used to identify paucibacillary (containing just a few bacteria) or extrapulmonary (outside of the lungs) TB.

   By taking advantage of the state of the art CRISPR/Cas research and combining it with phage diagnostics, we designed a sensor that is able to screen for a specific target gene. Our producer strain carries a helper plasmid that codes for the phage capsid proteins of M13 as well as our phagemid plasmid with our sensor system. Our sensor system consists out of three parts: the Cas9 gene under a constitutive promoter, the anti-KAN gRNA also under a constitutive promoter and the reporter element LacZ under the control of the pRecA promoter. As the helper plasmid doesn’t have a packaging sequence, only our sensor system phagemid is packed into the M13 capsids. As M13 is a lysogenic phage, the phagemid particles are exported into the media where they can be easily isolated and are ready to use to sense if an other strain contains our target sequence.

   Our system is highly modular as with the simple change of the gRNA target sequence (20bp), any gene of interest can be identified and targeted. This means that our system can be used for different applications and purposes, as for example a mutation screening. It is also not specified only for E.coli and the phage M13. Our whole system can be easily transferred to other phage systems that target other host bacteria. In our case, as we want to develop a sensor to test Mycobacterium tuberculosis, we could in principle transfer our system to mycobacteria phages. With what we are presenting here, we have a proof of concept in E.coli. By using host specific phages, we can easy identify specific bacteria and genes they carry. By building a library of phages with different gRNAs we could screen for many different genes in parallel including other resistances or pathogenic genes.

   The mechanism we are using for our sensor is based on the CRISPR/Cas System. The CRISPR/Cas genome editing method, which recently became very popular, is derived from the bacterial “immune system”. CRISPRs are Clustered Regularly Interspaced Short Palindromic Repeats that are part of the bacterial genome. These loci contain multiple short repeats. In between the repeats are so called spacers that are sequences derived from extrachromosomal DNA, e.g. from invading viruses or plasmids (Figure 4). Within bacteria, those CRISPRs are naturally used to detect and destroy foreign DNA that is saved in the spacers.

In total the CRISPRs and the spacers form a so-called CRISPR array that is transcribed as one unit. CRISPR associated proteins (Cas proteins) are involved in further processing and action steps of the CRISPR system. There are many different regulation systems, here we describe the Cas9 system that will be used for our purposes (Figure 4).

The transcribed mRNA is processed into so called crRNA (CRISPR RNA), which includes the spacer sequence of foreign DNA. Together with a transactingCRISPR RNA (tracrRNA), the crRNA forms a duplex that is cleaved by RNaseII. The resulting hybrid serves as guide for the Cas9 protein that generates double strand breaks at the position the RNAs guide it to (Figure 3). By this double strand break the invading DNA is destroyed. The last years, researchers discovered the CRISPRs as a method for genome editing.

Figure 4: CRISPR/Cas technology description (Addgene.org).

   Our reporter system consists out of the RecA promoter and LacZ. Originally, the RecA promoter of E.coli has its main role in the activation of the SOS repair system. It is regulated by the LexA repressor, which binds to the SOS box sequence of the promoter. DNA damage leads to an inducing signal, which then activates the RecA protein. In the SOS response the LexA repressor is cleaved by the RecA protein, so the full RecA expression can be reached. The RecA protein can then repair both single stranded and double stranded DNA breaks.

In our project RecA promoters used to detect double stranded DNA breaks in the region of antibiotic resistance genes caused by the CIRSPR/Cas system. To get the desired activation of the promoter, it is important to assure that no other stressing agents might activate the RecA promoter. Those agents could be UV light, X-ray, ionizing radiation and different types of DNA breaking compounds.

   Up to this point we cloned each of our parts into BioBrick vectors. We cloned the gRNA anti-Kan into pSB1C3 and pSB1A3.We used for this the assembly standard BioBrick cloning as well the new proposed Assembly Standard.. Both assemblies were successful and proved by colony PCR as well as by sequencing. We also used both standards for cloning the crRNA..

   For the characterization of the Cas9 and the gRNA anti-KAN we performed a killing assay . Therefore we co-transformed the Cas9 with gRNA anti-KAN into host bacteria that contain the KAN resistance gene cassette as well as without the cassette. In the KAN resistance bacteria, the CRISPR/Cas complex generated generate double strand breaks that killed the bacteria. After co-transforming the plasmids, we plated the bacteria on the one hand on the single antibiotic where the resistance is carried by the Cas9 plasmid as well as on the single antibiotic which resistance is present on the gRNA plasmid. As the two plasmids contain the same origin of replication, this will selesct for only the single plasmid and hence a non-functional CRISPR/Cas system. To get a working system, we plated the co-transformed bacteria on both antibiotics, of which the reistances were on the two plasmids. As we can see in Figure 6, if we select for both plasmids, we get a reduced number of colonies in the strain that contains the target sequence, the kanamycine resistance cassette. We attribute this reduced number to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency. This leads after repeatedly double strand breaks generated by the CRISPR/Cas system to cell death which explaines the reduced colony number.

  To characterize the pRecA –LacZ construct, we induced double strand breaks with Mytomycin C (MMC) and analyzed the resulting LacZ expression levels with the Miller assay. The same time, we wanted to characterize our final system: our target strain with the kanamycin resistance gene that also carries the reporter and the Cas9. The gRNA is delivered by a phage. As positive control, we used MG1655, which has a constitutive expression of LacZ, as negative control the parental strain of the target strain, which doesn’t have a kanamycin resistance cassette but also has the reporter and Cas9. As it can be seen in Figure 7, we didn’t get any expression of LacZ beside in the positive control, the MG1655 strain.

Figure 7: Miller assay Using the Miller assay to quantify LacZ expression levels in different strains: MG1655 which has a constitutive expression, the target strain + KAN, the control strain -KAN, with and without phages as well as the target strain only with the reporter, induced with Mitomycin C.

As previously mentioned, the reporter didn’t work as expected. This can have several reasons. As our other positive control (beside MG1655), the MMC induced strains carrying the reporter didn’t show any expression, it stands to reason that the reporter itself is not functional. Another reason could be, that the phagemid didn’t deliver the gRNA or not in enough amounts to show an effect of the complete working system.

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