When Francisco Mojica, a research scientist at the University of Alicante, Spain, set out to study genetic repeats in bacterial organisms, he had no idea his research would set the pace for discoveries that would have a profound impact in the fields of diagnostics and genetic engineering. The big discovery came in the early ’90s when Mojica first noticed the repetitive sequences, which he later named “short regularly spaced repeats” or SRSRs, while studying Haloferax mediterranei, a type of archaea found in salt flats.

In 2001, after a decade of research that found similar sequences across a plethora of bacteria and archaea, Mojica and another researcher coined the term “CRISPR,” which stands for clustered regularly interspaced short palindromic repeats. The goal was to unify further research using a consistent terminology, as a research group led by another scientist, Ruud Jansen was using SPacers Interspersed Direct Repeats (SPIDR) to describe the same phenomenon. The naming convention was agreed, but neither group had a firm grasp on the significance of their findings.

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 acidbased 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 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 finetuned 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 choosing the perfect tool from a toolbox – each has its own unique abilities. Even with a molecular GPS there’s a certain degree of amplification required to maximise the accuracy of a diagnostic test. The choice of amplification technology in a CRISPR-based diagnostic assay depends on various factors, including the specific target, the intended application, and the desired level of sensitivity and specificity. “It can be hard to select an appropriate amplification technology, pair it with a CRISPR Cas enzyme that works in realtime at the given amplification temperature, and design both amplification primers and a CRISPR guide to detect the desired DNA and RNA target with the highest sensitivity and specificity,” notes Dechario. “But, AI algorithms can accomplish all these tasks swiftly and efficiently, in silico, typically within hours.”

Legacy diagnostics have historically been unable to meet the demand for decentralised testing due to a trio of historical barriers: cost, convenience, and test performance. “Traditional diagnostic approaches require leaving work and home to visit a physician and wait for results, but with CRISPR, that can be done in a matter of minutes at home,” Dechario explains. “For events that require urgent public health interventions in low-resource settings — such as a field response to an emerging disease — current diagnostics can be too expensive and slow to process enough tests to control an outbreak, for example. Now, advances in CRISPR diagnostic technology can make rapid tests for a range of infectious diseases available to low and middleincome countries.”

An exciting future?

Like any other emerging technology, the CRISPR-Cas system has challenges. One significant drawback is its tendency for off-target effects – non-specific binding on the organism’s genome. To mitigate this, specialised bioinformatic tools can enhance precise guide RNA design selection. According to Davies though, these safety and ethical questions that have clouded CRISPR in the past 5-6 years have focused on “germline applications” – gene editing practices that apply to reproductive cells, where any modifications would be passed on to offspring. “They do not apply to developing lab or homebased diagnostic tests,” he says. What is a relevant challenge for those developing CRISPR-based diagnostics, is making a product hospitals want to use. “These systems show great promise for the future but must prove that they outstrip existing diagnostic procedures to become mainstream tools in the diagnostic industry,” Davies adds.

For Dechario, gaining this acceptance among clinicians is the major challenge to overcome if patients are to benefit from CRISPR-based diagnostics. “In clinical settings, the challenge is the clinical community embracing the change in patient flow,” he says. “Rather than a patient coming in looking for a diagnosis, with CRISPR decentralised diagnostics – patients will more frequently arrive with a diagnosis for reflex testing and treatment,” says Dechario. “Upon regulatory clearance, it will be critical for the medical community to accept these diagnostic results as equivalent to what they would have traditionally been ordered from central labs after a patient visit.”

Regarding the future of diagnostics, Dechario sees the decentralising of tests, driven by new products leveraging CRISPR, as being key to improving global health and eliminating healthcare inequities. “This month, we announced funding from the Bill & Melinda Gates Foundation to advance rapid, instrument-free molecular diagnostics for Human Papillomavirus (HPV),” he says. “Of individuals with this condition, 81% live in low and middle-income countries and lack access to essential diagnostics — as such — there is a global opportunity to bring decentralised testing to these patient populations by engineering diagnostics through synthetic biology so we can bridge the gap in delivering life-saving medicines.” Davies is likewise excited for the future of medical science as it pertains to the use of CRISPR as a diagnostic tool – especially if it means we’re better prepared for another virus like SARS-CoV-2. “With two biotech companies, Sherlock and Mammoth, leading the way in driving the development of CRISPR diagnostics, I expect to see more exciting advances in the future – hopefully in time for the next pandemic.”