Team:CMUQ/Design
CRISPR-Cas12a: a novel biotechnology replacement to carrier testing
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CRISPR-Cas12a (Cpf1) 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 completely degrade both linear and circular ssDNA molecules within minutes. To date, this technology has been used as a genome editing tool; we propose the use of CRISPR cas12a for various diagnostic purposes, specifically for carrier testing to detect heterozygosity in autosomal recessive diseases.
The indiscriminate degradation of ssDNA will be used to screen for carriers of Sickle Cell Anaemia, one of the most common severe monogenic disorders in the world. We will use saliva samples as source of DNA. Target DNA will be amplified by RPA to make it increase the chances of Cas12a finding and binding onto the target, unleashing indiscriminate cutting of single-stranded DNA. This includes the degradation of DNA attached to a fluorescent marker , which will separate the suppressor from the fluorescent molecule which can then be detected by the naked eye. The test will take about 30 min and will be able to replace expensive, time consuming, laboratory and labor intensive methods used currently. This moves us to a field-ready diagnosis technique, that can be used anywhere and custom designed to target any mutation.
CRISPR-Cas12a: a novel biotechnology replacement to carrier testing
- Up until today, we often hear about how CRISPR technology is revolutionizing medicine in many ways, yet mostly as a genome editing tool. This year, our team proposes the usage of CRISPR-Cas12a for diagnostic purposes, in particular, for carrier testing to detect heterozygosity in autosomal recessive diseases. CRISPR-Cas12a (Cpf1) proteins are DNA targeting enzymes that cut and bind DNA depending on an embedded guide-RNA sequence designed to match a targeted strand of the manipulated DNA. The binding of the guide- RNA sequence with the DNA initiates a non-specific single-stranded DNA (ssDNA) cleavage activity in Cas12a, this is sufficient to completely degrade both linear and circular ssDNA molecules within minutes. The indiscriminate degradation of ssDNA will be mainly used to screen for carriers of Sickle Cell Anaemia, one of the most common severe monogenic disorders in the world. Nevertheless, with minor changes, this technique can be custom designed to target any mutation. Using saliva as the DNA source, the target DNA will be amplified by RPA to increase the chances of Cas12a in finding and binding onto the target strand unleashing indiscriminate cutting of single-stranded DNA. This process, nevertheless, includes the degradation of DNA attached to a fluorescent marker , which will be exposed and detected by the naked eye when the the suppressor gets detached from the fluorescent molecule. This thirty minutes testing method can not only replace expensive, time consuming, laboratory and labor intensive methods used currently, but it can serve as an introduction to field-ready diagnosis technique which can be used anywhere.
Sickling Test
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Item
Sickling Test
Effectivity
01
Specimen
3ml in EDTA tube(may be part of CBC)
02
Transport Temperature
Ambient ref
03
Days test is performed
Daily
04
Turnaround Time
Two working days
05
Method
Sickling test may be done alone or is done as a part of HB electrolysis
06
Reference Value
Negative
07
Interpretation
Positive Sickling Test occurs in HB S disease, sickle cell trait and HB S/C double heterozygosity. The test is done to confirm sickling or any time when peak is detected by HPLC