Diagnostics
That was until advances in the field of genomic sequencing led to more data on different organisms, including DNA sequences related to CRISPR. Mojica had found in previous research on E. coli that the DNA sequences in the CRISPR locus matched those of a virus known to infect the bacteria. With a wealth of genomic data now available, he and other researchers were able to find DNA sequences for a range of bacteria. The eureka moment came when they found matches between sequences in the CRISPR locus of these bacteria and pathogens known to infect them, just like Mojica had earlier with E. coli. With these findings, a new hypothesis was born: CRISPR forms part of an adaptive immune system that captures snippets of viral DNA and stores them as irregular sequences to protect against subsequent attacks. Each sequence is transcribed into a long RNA molecule known as pre-CRISPR RNA (pre-crRNA), which is processed into smaller individual crRNAs, each containing information about one specific viral DNA sequence. Armed with this immunity, if a bacteria or archaea is invaded by a virus for a second time, the crRNA acts as a guide for CRISPR-associated (Cas) proteins, which bind to DNA sequences that match the crRNA and launch an attack of their own, selectively cleaving it or blocking the action of enzymes – both of which prevent replication. It’s this ability to cleave specific parts of DNA that led to the breakthrough research proving that crRNA could be programmed to snip and introduce changes to sequences, potentially curing or at least leading to new treatments for genetic diseases. But while research in this area is yet to reach patients, today, nucleic acid- based diagnostics that leverage CRISPR are rapidly expanding, helping clinicians detect a wide range of molecular targets.
“CRISPR-based approaches can address the need for decentralised diagnostics and improve access to molecular detection worldwide – making it easier to identify single unique nucleic acid sequences on a global scale in a cost-effective manner,” explains Bryan Dechario, CEO of Sherlock Biosciences, one of two major companies founded to develop CRISPR into medical products. “Cas enzymes have also demonstrated exceptional effectiveness, even in the face of crude sample preparation techniques, simplifying the creation of accessible diagnostic testing platforms.”
Infectious and non-infectious disease detection
The fast-evolving field of CRISPR-based detection builds on the specificity, programmability, and usability of CRISPR technology to develop nucleic acid-based diagnostic models. It also aids in multiplexing — the simultaneous detection of multiple diagnostic targets in a single reaction, resulting in a lower per-target cost. A perfect example is the second version of specific
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high-sensitivity enzymatic reporter unlocking developed by Sherlock Biosciences, also known as SHERLOCKv2. The technology enables the detection of about four targets in one multiplexed reaction, detecting distinctive pathogenic nucleic acid signatures in a sample using the company’s proprietary ‘smart amplicon detection’. Upon signature detection, the cell activates the CRISPR Cas enzyme to generate a robust signal on multiple channels, readable via a simple paper strip, in-lab device, or mobile-phone-accessible electrochemical readout.
During the covid-19 pandemic, Sherlock Biosciences invented the first commercially available CRISPR-based diagnostic assay for SARS-CoV-2 as a spin-off of the SHERLOCK-based detection system. The company received emergency use authorisation from the United States Food and Drug Administration (FDA) for a modified SHERLOCK-based two-step assay in May 2020. Two years later, rival company Mammoth Biosciences followed in similar footsteps and secured FDA approval for its high-throughput Covid test – the DETECTR. By employing Cas12a, researchers fine- tuned the SARS-CoV-2 DETECTR assay into a swift two-step process. This optimised method detects the N (nucleoprotein) and E (envelope small membrane protein) genes of the SARS-CoV-2 genome alongside a human control gene (the ribonuclease P protein). The process unfolds in under 40 minutes with a simple nasopharyngeal swab.
The pandemic may have been a catalyst for the development of diagnostics, but speak to Kevin Davies, and it’s hard not to get excited about the prospects of nucleic acid-based detection outside of Covid-19. “CRISPR diagnostics offer several potential benefits in accuracy, speed, and affordability,” stresses Davies, executive editor of The CRISPR Journal. “There have been significant advances and numerous published reports in the application of CRISPR to developing diagnostic methods for Covid-19 and other infectious diseases.” According to Dechario, advances in CRISPR are not limited to common respiratory infections like Covid-19, flu and RSV. “Recent breakthroughs reveal CRISPR’s ability to detect subtle mutations and facilitate real-time quantification of DNA and RNA, opening up exciting avenues for prenatal testing and cancer detection,” he says.
Developing CRISPR-based diagnostics The benefits CRISPR can have in the field of diagnostics are clear, but how simple is it to develop a CRISPR-based diagnostic? The answer is not very. First, the specific genetic sequence of the target disease must be identified, which is a bit like finding unique clues in a complex puzzle. Crafting the guide RNA is akin to devising a molecular GPS and directing the CRISPR enzyme, be it Cas9 or Cas12, wherever it’s needed. Selecting the suitable Cas enzyme is like
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