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Team:Paris Bettencourt/Project/Detect
From 2013.igem.org
Background
CRISPR/Cas systems generate site-specific double-strand breaks and have recently been used for genome editing.
Results
- Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks.
- Testing the new assembly standard for our cloning.
- Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette.
BioBricks
Aims
Building a genotype sensor based on CRISPR/Cas that reports on the existence of an antibiotic resistance gene.
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Aim
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.
Figure 1: Detection and reporting of an antibiotic resistance gene with a CRISPR/Cas system.
Motivation and existing TB sensors/tests
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.
Diagnosis methods using culture methods require laboratory infrastructure that is not widely available in countries with a high burden of TB and results are only available after a few weeks.
Other conventional methods used to diagnose multidrug-resistant TB (MDR-TB) also rely on the culturing of specimens followed by drug susceptibility testing (DST). Results take weeks to obtain and not all laboratories have the capacity to perform DST of first-line or of second-line drugs.
MTB/RIF is a new rapid molecular test that can diagnose TB and rifampicin-resistant TB within hours. Molecular tests, such as GeneXpert, unlike culture-based methods, are fast, accurate and can detect drug-resistant strains. But the high costs and need for laboratories make access an issue for developing countries.
The new method, published in the Journal of Applied Microbiology, uses a microcalorimeter to detect heat produced by Mycobacterium tuberculosis, the bacterium that causes TB, on a growth medium. The study showed that detection takes 4–5 days but more sensitive microcalorimeters could detect tuberculosis in 24 hours.
System Design
To use our system, we need two strains, a phagemid producing strain and the target strain. The producer strain contains a helper plasmid, which produces the capsid proteins for the phage and the phagemid plasmid with the sensor elements, which is packed up into the M13 capsids. The helper plasmid encodes for everything of the phage but doesn’t contain the packaging sequence, while the phagemid plasmid only contains the packaging sequence but doesn’t code for anything else of the M13 phage. As M13 is a lysogenic phage, the phagemid particles are exported into the media, where they can be collected and transferred to the target strain. The phagemid infects cells that are conjugating, as M13 needs a sex pili to attach to the cell and release the plasmid plasmid into the cell.
Within the target strain, the Cas9 protein, as well as the gRNA get expressed. The gRNA then attaches to the Cas9 protein and guides the Cas9 to the target sequence (Kanamycin resistance gene). Once bound to the sequence, the Cas9 protein generates a target specific double strand break.
This double strand break leads to the cleavage of LexA. LexA is a protein bound to the pREC promoter to inhibit the expression of genes under the control of the pREC promoter. With the cleavage of LexA, the expression of our reporter LacZ is started, which leads in the presence of xgal to a blue color output.
Figure 2:Course of antibiotic resistance detection and reporting Title
Background
CRISPRs
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 3). 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 3).
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 3: CRISPR/Cas technology description (Addgene.org).
By designing the spacer sequence, specific sequences in the genome can be targeted. With the provision of a sequence with homologous sequences, easy insertion can be done. This method can be used to insert mutations or new sequences nearly everywhere in the genome.
Recently many papers have been published for genome editing in bacteria (Jinek et al., 2012, Jiang et al. 2013), yeast (DiCarlo et al. 2013) and mammalian cells (Mali et al., 2013, Cong et al., 2013). They describe how to use the CRIPRs for different purposes. One requirement to target a sequence is a NGG at the end of the sequence. This NGG sequence is called PAM - protospacer adjacent motif, while the target sequence itself is called the protospacer, which should have a length of around 12 to 20 bp.
In our approach we test two different systems, the systems of DiCarlo et al. 2013 and Jiang et al. 2013. The system of Jiang is a system developed for bacteria and especially for E.coli. The system of DiCarlo is based on a paper of Mali et al. 2013 and adapted to yeast.
Different from the Jiang paper, the DiCarlo paper uses a gRNA (guideRNA) to guide the Cas9 (Figure 4). The gRNA is actually the RNA that results after transcribing and folding is the same complex as you get after the processing of the tracrRNA with the crRNA. It is hence an improvement, which makes the design and expression easier. But as the system hasn’t been tested before in bacteria, we will use both systems to make sure we get the desired results.
Figure 4:Cas9 protein interacting with CRISPR gRNA
RecA promoter
Our reporter system consists out of the RecA promoter and LacZ. Originally, the RecA promoter of Escherichia 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 r
