Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains one of the world's most serious public health problems. Although tuberculosis is curable and the treatment success rate is high, it is still the second most common cause of death from infectious disease. Most of the deaths occur for the lack of effective identification of those in need of therapy. Case detection is currently the rate-limiting step in TB control.
The currently widely-used TB detection methods all have their own problems. Nucleic Acid Detection (NAD) is a safer, faster, and more sensitive detection method. However, its shortcoming is critical: the high false-positive rate from non-specific amplification, and the requirement of extremely expensive clumsy instruments; these make NAD not common for TB diagnosis.
To obviate such shortcoming, Peking iGEM developed a novel detection system, paired dCas9 (PC) reporter, that converts the sequence-specific information of pathogenic bacteria's genome (in our case, M. tuberculosis, MTB) into easily readable signal including bioluminescence, pigment, or electric current. Our PC Reporter was successfully applied to the detection of real pathogenic M.Tuberculosis H37Rv (isolated genomic DNA that is absolutely safe, prepared by our collaborator, not by us). Combined with our work in multiple-marker array (using MTB-specific markers extracted from the entire genome of MTB) and hardware development, our PC reporter system is expected to be a powerful tool in TB diagnosis, with a huge potential for various applications and extensions.
It is well known that CRISPR/dCas9 has a unique ability to be programmed to bind any sequence with the assistance of sgRNA; it was conventionally used for DNA editing or genome study. In our project, however, we integrate split reporters into CRISPR/Cas9 by translationally fusing two fragments of a split reporter to dCas9, respectively, to convert the sequence-specific information of pathogenic bacteria's genome (in our case, M. tuberculosis) into easily readable signal including bioluminescence or pigment. We demonstrated that the PC reporter is highly compatible with NAD-based diagnosis using isolated genomic DNA of MTB.
To deal with challenges from clinical practices, such as strain mutations, sample variations, and other uncontrollable environmental factors, we designed an array to extract sequence information from the entire genome of M. tuberculosis, for our PC Reporter to measure multiple sites on the target genome at one time, thus to improve the reliability of diagnosis. By statistics analysis, we are able to present the readouts in a quantitative way.
Noticing that most TB cases occur in developing areas, we built an electronic device (despite prototype), which was portable, affordable, and easy to use for local medical workers.
In order to popularize our diagnosis method in developing areas, we invented two novel isothermal amplification methods whose mechanism and operation requirements have been significantly simplified.