Cas-Find is a point-of-care diagnostic tool using the CRISPR-Cas9 system with the potential to radically change the speed of disease diagnosis, with a particular focus on the detection of sexually transmitted infections (STI’s).
Sexually transmitted infections (STIs), including those caused by the human immunodeficiency virus (HIV) types 1 and 2, remain an important focus area for global public health. This is due to the high morbidity associated with STIs, such as the sequelae of reproductive tract infections, cervical cancer, congenital syphilis, ectopic pregnancy and infertility, as well as the morbidity of HIV-related illness and death from acquired immunodeficiency syndrome (AIDS).
More than 30 bacterial, viral, and parasitic pathogens are transmissible sexually and constitute a group of infections referred to as sexually transmitted infections (STIs). Although some of the pathogens can be acquired through routes other than sexual transmission, epidemiologically, sexual contact is more important for their transmission from one person to another (See Table 1 for information on the main sexually transmitted pathogens).
http://hai-sti.roche.com/images/hai-and-sti-epidemics.png
Point-of-care testing (POCT): ‘tests designed to be used at or near the site where the patient is located, that do not require permanent dedicated space, and that are performed outside the physical facilities of the clinical laboratories’ i.e. testing at the time and place of patient care (Santrach 2007).
With the aim of POCT to bring the test conveniently and immediately to the patient it increases the likelihood of the physician and patient receiving results more quickly and allowing immediate clinical management decisions to be made. Rapid decision making, reduced number of outpatient clinic visits, reduced number of hospital beds required and occupied, overall ensuring the optimal use of professional time.
When used for diagnosis, the time required for test results to become available to guide management should be considered when choosing which test to use, since infected persons may transmit infections to others, may suffer complications of infection, or may be lost to follow-up in the interval between testing and notification of test results
Laboratory-based tests, such as culture or nucleic acid amplification testing, may require special methods of specimen transport and specialized equipment and procedures for optimal performance, thus delaying the availability of results for immediate management decisions. Therefore laboratory-based diagnosis of STIs tends to be expensive in terms of equipment, reagents, infrastructure, and maintenance. Even more importantly, particularly in resource- constrained settings, the levels where most patients with STIs are processed have no laboratory facilities. (Peeling et.al. 2006)
In countries with a high burden of sexually transmitted infections (STIs), where laboratory services for STIs are either not available or limited services are available and patients may not be able to physically access or to pay for these services.
Currently in many high risk 3rd world countries the World Health Organisation (WHO) recommends the use of syndromic management where patients are treated for all the major causes of a particular syndrome. Syndromic management of STIs works well for urethral discharge, pelvic pain, and genital ulcer disease, but evaluations of the WHO flowcharts have shown that the algorithm for vaginal discharge lacks both sensitivity and specificity for the identification of women with Chlamydia trachomatis and Neisseria gonorrhoeae infection. Simple, affordable, rapid tests that can be performed at the point-of-care (POC) and enable treatment and case management decision to be made are urgently needed for these infections (Unemo 2013). Simple rapid POC tests are needed not only to increase the specificity of syndromic management, and reduce over-treatment of genital gonococcal and chlamydial infections but also to screen for asymptomatic STIs. A mathematical model estimated that a test for syphilis that requires no laboratory infrastructure could save more than 201,000 lives and avert 215,000 stillbirths per year worldwide. A similar test could save approximately 4 million disability- adjusted life years (DALYs), avert more than 16.5 million incident gonorrhoea and chlamydial infections and prevent more than 212,000 HIV infections per year (Unemo 2013).
A = Affordable
S = Sensitive
S = Specific
U = User-friendly (simple to perform in a few steps with minimal training)
R = Robust and rapid (can be stored at room temperature and results available in <30 minutes)
E = Equipment-free or minimal equipment that can be solar- or battery-powered
Yes! Many are in current circulation, some examples being:
Current Principles of rapid point-of-care test technologies for STI’s:
Lets take one of the most common sexually transmitted infections in the UK affecting on average 200,000 people each year, chlamydia (PHE 2015). Currently to test for this infection you take either a urine sample or swab to determine if the chlamydia bacteria (Chlamydia trachomatis) is present. Analysing the samples involves using nucleic acid amplification tests (NAAT) such as PCR or by growing a chlamydia culture. Cultures incur high costs, have varying sensitivity and limitations for widespread screening whilst both methods are laborious and often time consuming. Cas-Find aims to reduce the time to disease detection. Using the CRISPR-Cas9 system specific sequences on the suspect bacteria can be immediately detected, emitting a measureable fluorescence if present. This point-of-care nature of our project can significantly advantage those where lab access if limited, expensive and the speed of diagnosis is crucial in making the appropriate clinical decisions.
|
Pathogen |
Clinical manifestations and other associated diseases |
|
Bacterial infections |
|
|
Neisseria gonorrhoeae |
GONORRHOEA |
|
Chlamydia trachomatis |
CHLAMYDIAL INFECTION |
|
Chlamydia trachomatis (serovars L1–L3) |
LYMPHOGRANULOMA VENEREUM |
|
Treponema pallidum |
SYPHILIS |
|
Haemophilus ducreyi |
CHANCROID |
|
Klebsiella (Calymmatobacterium) granulomatis |
DONOVANOSIS (GRANULOMA INGUINALE) |
|
Mycoplasma genitalium |
Men: urethral discharge (nongonococcal urethritis) |
|
Viral infections |
|
|
Human immunodeficiency virus (HIV) |
ACQUIRED IMMUNODEFICIENCY SYNDROME (AIDS) Both sexes: HIV-related disease, AIDS |
|
Herpes simplex virus type 2 Herpes simplex virus type 1 (less common) |
GENITAL HERPES |
|
Human papillomavirus |
GENITAL WARTS |
|
Hepatitis B virus |
VIRAL HEPATITIS |
|
Cytomegalovirus |
CYTOMEGALOVIRUS INFECTION |
|
Molluscum contagiosum virus |
MOLLUSCUM CONTAGIOSUM |
|
Kaposi sarcoma associated herpesvirus |
KAPOSI SARCOMA |
|
Protozoal infections |
|
|
Trichomonas vaginalis |
TRICHOMONIASIS |
|
Fungal infections |
|
|
Candida albicans |
CANDIDIASIS |
|
Parasitic infestations |
|
|
Phthirus pubis Sarcoptes scabiei |
PUBIC LICE INFESTATION SCABIES |
Peeling, R.W., Holmes, K.K., Mabey, D. (2006) Rapid tests for sexually transmitted infections (STI’s): the way forward. Sex Transm Infect. 82:1-6.
Public Health England. (2015) Sexually transmitted infections and chlamydia screening in England. Infection Report. 10:22
Santrach, P.J. (2007) Current and Future Applications of Point of Care Testing. Mayo Clinic. Access online at: http://wwwn.cdc.gov/cliac/pdf/addenda/cliac0207/addendumf.pdf
Unemo M. (2013) Laboratory diagnosis of sexually transmitted infections, including human immunodeficiency virus. WHO.
Laboratory-based tests, such as nucleic acid amplification (NAA) or culture, require special methods of specimen transport, alongside specalised equipment and procedures for optimal performance [2]. As such the utilisation of laboratory-based tests is generally expensive in terms of equipment, reagents, infrastructure and maintenance. This limits the availability of results for immediate use in management decisions, potentially impacting on patient prognosis. In addition the majority of STI testing is conducted in resource-constrained environments, where such laboratory facilities are unavailable [3].
Fig 1. Summary of Cas-Find project
Dr. Daniel Pass designed sgRNA constructs targeted to the E. coli 16S rRNA locus in order to facilitate proof-of-concept testing of our in vitro system. The design of these constructs was achieved using a Python script developed by Dr. Pass to test FASTA formatted genomic DNA for paired target sequences using guidelines from Takara Bio USA alongside additional sources. This script initially identifies a proto-spacer adjacent motif (PAM) sequence (5'-NGG-3') in this FASTA sequence. The sgRNA sequence is complementary to the 20 nucleotides upstream of the PAM sequence. This is passed to BLASTn to test for simple alignment against the reference dataset, which could include the remainder of the species genome, or multiple cross-reactive species. The output is a FASTA table of potential probes, and a table.txt file of the same information in a graphical representation.
Fig 2. Summary of sgRNA design
Fig 3. Summary of Characterisation
As a link to our use of luciferases in the Cas-Find project we developed an interest in biological imaging. This was largely influenced by the research of our Secondary PI, Amit Jathoul in this general area.
One challenge in the field of biological imaging is in the use of fluorescent and bioluminescent proteins that emit light with longer wavelengths. The mKeima RFP variant has been previously used as a long stoke shifted fluorophore and was the subject of the SPECTRA project of the 2013 UCL iGEM team .
Therefore as a side project we planned to alter the potential usage of the Lux Operon biobrick (Cambridge iGEM 2010 BBa_K325909) by inducing a red-shift in its bioluminescence via an interaction with the mKeima protein.
We hoped to show that blue light produced by E.coli expressing the native LUXoperon would excite the mKeima protein resulting in the emission of red light.
We aimed to analyse the interaction between LUXoperon and mKeima in two ways:
The mKeima protein has been added to the registry by the UCL_PG team of 2013 (BBa_K1135001). However as part of this side project we have submitted two unique parts to the registry that contain this protein:
We aimed to grow these biobrick-containing bacteria in the presence of arabinose to induce expression of each construct and then measure the fluorescence and bioluminescence of the bacteria to assess:
Initially we sub-cultured overnight bacterial cultures (1/10) and grew for 3hours prior to induction of gene expression by addition of arabinose at various concentrations for approximately 6hours.
Following induction we used a Cary XXXX to measure fluorescence and bioluminescence across appropriate spectra. The LUXoperon has been previously shown to emit light at 488nm whilst the emission wavelength of mKeima is 620nm. We also used a non-biobrick construct expressing pBAD::sfGFP as a control, for which the emission wavelength is 510nm.
Firstly we grew biobrick-containing E.coli overnight and after sub-culturing (1/10) grew for 2hr before induction with mM concentrations of arabinose.
Figure 1 shows that at these concentrations (5-20mM) of arabinose, bacteria containing the native LUXoperon showed a predicted bioluminescence spectra. However none of the other bacteria were bioluminescent.
Figure 2 shows that at these concentrations (5-20mM) of arabinose, bacteria containing sfGFP showed a predicted fluorescent spectra. However none of the other bacteria showed any fluorescence across the range of tested wavelengths.
Previous work has demonstrated that mKeima expression under the pBAD promotor was best at lower concentrations of arabinose. Therefore we grew bacterial sub-cultures for 6hr in 100uM and 250uM Arabinose (Figure 3 and 4). Unfortunately this did also not show bioluminescence or fluorescence that was suggestive of mKeima expression.
Finally we attempted to stimulate expression of the mKeima-containing biobricks BB_K2060001 and BB_K2060002 by growing bacterial cultures overnight and spiking in arabinose at 10mM for 2hr before measurement.
Figure 5 demonstrates that the LUXoperon-mKeima gave a bioluminescent blue-light output, indicating that the LUXoperon is working correctly. However figure 6 shows that there is no inducible fluorescence generated by these bacteria. This indicates that further work is necessary to stimulate effective expression of both the LUX components and mKeima from the same operon.
iGEM headquarters recently published a paper demonstrating the challenges in expression of fluorescent proteins, BB_K2060001 or BB_K2060002. Sadly we ran out of time to conduct further analysis but might would recommend the following experiments:
We hope that future iGEM teams will take advantage of the availability of these biobricks as potentially very useful tool for the analysis of gene expression. With increased time the development of these tools will be of benefit for the entire iGEM community.
