Team:CMUQ/Background
CRISPR-Cas12a: a novel biotechnology replacement to carrier testing
Cas12a - Recognizing Carriers of recessive traits to save generations
Sickle Cell Anaemia is one of the most common, severe, monogenic disorders in the world. Genetic studies indicate that this condition is inherited in an autosomal recessive pattern where parents, often showing no symptoms of the condition, each contribute one copy of the mutated gene. This genetic disease is mainly caused by rs334, which is a single nucleotide polymorphism (SNP) located in the Hemoglobin-coding gene (HBB). Our approach overcomes the limitations of sequencing, it being a cost-ineffective, labour-intensive, and location-specific method. Utilizing CRISPR for purposes other than gene editing has allowed us to create a novel, field-ready, diagnostic technique for carriers of recessive traits. Cas12a proteins are DNA targeting enzymes that recognize DNA based on a guide RNA sequence designed to match a target. The binding initiates non-specific single-stranded DNA (ssDNA) cleavage activity in Cas12a sufficient to degrade linear and circular ssDNA within minutes. Through this, ssDNA attached to fluorescent dye and quencher, serving as reporters, will undergo degradation. Upon cleavage, the quencher is released and fluorescence is emitted. We designed, built and programmed a hand-held device that can detect the fluorescence with high sensitivity. Simply, DNA obtained from saliva sample, inserted into the device, diagnoses carriers of Sickle Cell Anemia.
Our Project
In Middle Eastern populations, many carriers are uaware of the mutations that they can pass on to their children and the probability of their child being affected. To address this issue, we endeavored to create a reliable, inexpensive test to help with family planning and care. Our project utilises CRISPR-Cas12a (Cpf1) as a diagnostic tool for carriers. Cas12a proteins are DNA targeting enzymes that bind and cut DNA based on a guide RNA sequence that is designed to match a target. RNA-guided DNA binding initiates non-specific single-stranded DNA (ssDNA) cleavage activity in Cas12a sufficient to degrade both linear and circular ssDNA molecules within minutes. When the guide RNA matches the mutation in the patient sample, there is as indiscriminate degradation of ssDNA with a quencher and fluorescent dye. Upon cleavage, the quencher is released and the fluorescence is emitted. We designed, built, and programmed a hand-held device that can detect the fluorescence with high sensitivity. DNA samples will be extracted from saliva sample via a field-ready extraction kit, and by inserting the DNA solution into device, individuals are able to identify whether or not a genetic mutation is present.
We hope that this technique will make genetic testing and counselling more accessible, as we raise awareness of preventive medicine and aim to save generations.
Rees, D. C., Williams, T. N., & Gladwin, M. T. (2010). Sickle-cell disease. The Lancet, 376(9757), 2018-2031. doi:10.1016/s0140-6736(10)61029-x