Automated, high-throughput sequencing systems (above) have made an indelible impact on life sciences research. They are now poised to transform molecular diagnostics.Credit: Martin Krzywinski/SPL/Getty Images
CRISPR gene editing and next-generation sequencing (NGS) have transformed clinical and life sciences research. CRISPR permits the selective targeting and editing of specific nucleotide sequences, with a degree of accuracy and ease unachievable just a few years ago. NGS allows for high-throughput, precise and affordable DNA or RNA sequencing.
Together, the technologies have fostered numerous advances in basic molecular and cell biology and disease research; the discovery of CRISPR even earned the researchers behind it a Nobel Prize in 2020. They are also aiding in the development of new gene and cancer therapies, such as CAR-T cells.
While those achievements are widely recognized, the impacts of CRISPR and NGS on molecular diagnostics could be just as profound. A few diagnostics based on CRISPR and NGS have already reached the clinic, but the number of applications in development is far greater. Those diagnostics could enable early disease detection, accurate disease monitoring and more agile administration of precision therapies across numerous fields.
As in the early days of immunosorbent assays or fluorescence in situ hybridization, CRISPR and NGS present significant opportunities for those developing molecular diagnostics. Where does the science stand, and where might it lead?
CRISPR as a diagnostic tool
In 2016, the Zika outbreak in the Americas set the stage for the first approved CRISPR diagnostic1. Since then, CRISPR diagnostics have been used to detect the Lassa and Ebola viruses, along with SARS-CoV-22,3. Infectious disease outbreaks have driven development thus far. But any setting that requires high selectivity of a known genetic target, such as the analysis of tumour samples, identification of inherited genetic variants or non-invasive prenatal testing (NIPT), is a possible candidate for CRISPR diagnostics4.
CRISPR is best known as a gene editing system in which a Cas9 protein is paired with a guide RNA engineered to complement a target DNA. When the target DNA is identified, Cas9 cleaves the sequence at a defined site, permitting gene excision or insertion.
The mechanism behind CRISPR-based diagnostics is similar to gene editing, in that it uses an engineered guide RNA. But it typically employs different Cas proteins, namely Cas 12, Cas 13 and Cas 14. Those proteins can be modified to generate fluorescent signals in the presence of a target nucleotide sequence, whether DNA or RNA, making the system suitable for assays or imaging.
While most researchers develop their own CRISPR diagnostics, two commercial platforms have recently arisen to speed the process. SHERLOCK was developed by Feng Zhang’s group at the Broad Institute, and DETECTR was created by Jennifer Doudna, who received a Nobel Prize for the discovery of CRISPR-Cas9, along with her team at the University of California, Berkeley5,6. Both platforms use CRISPR to offer rapid in vitro identification of target sequences at attomolar sensitivity.
As with any molecular diagnostic, CRISPR tests need sufficient target DNA for detection. For that reason, they often rely on an amplification step before detection. That is most frequently accomplished through polymerase chain reaction (PCR), a tried-and-true method that requires thermocyclers to amplify nucleotide sequences. Unlike true PCR-based diagnostics, such as reverse transcriptase PCR (RT-PCR), CRISPR diagnostics can use other amplification methods, too, which presents an advantage.
“When you start thinking about doing this in a clinician’s office, or in the field, they don’t have thermocyclers,” says Matthew Poling, Product Manager for Genome Editing products at Thermo Fisher Scientific. “For these diagnostics to move into patient-centric point-of-care work, LAMP is becoming more promising.”
LAMP, or loop-mediated isothermal amplification, is an emerging technique that permits the amplification of nucleotide sequences at a constant temperature. Rather that separating double stranded DNA with heat, as in PCR, LAMP requires a DNA polymerase, such as EquiPhi29 or Bsm DNA polymerase, to actively ‘unzip’ and amplify double stranded DNA. The technique can also be adapted to detect RNA sequences.
Along with CRISPR reagents, Thermo Fisher Scientific offers various LAMP polymerases as lyophilization-compatible enzymes. These lyo-ready enzymes are glycerol-free and remain stable when shipped and stored, making them better suited for use in the field or at the point-of-care.
Using CRISPR for cancer diagnostics
Among the clinical fields that might benefit from CRISPR diagnostics, oncology is perhaps the largest. While it’s not yet possible to walk into a doctor’s office and ask for a CRISPR test, researchers are working on it. In a proof-of-principle experimental setting, scientists used the SHERLOCK platform to detect known cancer mutations5. Others are taking different approaches.
Harveer Dev, a clinical lecturer and group leader in the Early Detection Programme at the Cancer Research UK Cambridge Centre, is currently working to analyse and identify prevalent biomarkers in prostate cancer. He is developing a CRISPR-based platform called ProCASP that can map genetic variants in individual tumours to help predict their susceptibility to certain drugs.
“The aim of it,” Dev says, “is to map the functional contribution of specific genes within an individual patient’s tumours.” Dev says that he hopes his work will eventually translate into a diagnostic that informs a patient’s treatment plan.
“If there were another layer of information that we could add to that,” he says, “we think that's going to be quite important in shaping the specific treatment that individual patients receive.”
In cancer and elsewhere, CRISPR diagnostics still face challenges. For example, current CRISPR diagnostics cannot yet be leveraged in a high-throughput setting. Also, all CRISPR-based diagnostic platforms require a single known target sequence, which can be particularly challenging in certain cancers and other multifactorial inherited disorders. Until CRISPR tools have advanced to be applied to multiple genes at once, diagnoses that require this type of multiplexing may be more suited to next-generation sequencing tools.
Next generation sequencing in diagnostics
Like CRISPR, next-generation sequencing (NGS) technologies have quickly advanced from a standard research application to a complex diagnostic tool.
“When NGS became affordable, it enabled hypothesis-free experiments, where you don't need to know what you're looking for,” says Žana Kapustina, R&D manager at Thermo Fisher Scientific in Vilnius, Lithuania.
Today, rapid parallel sequencing enables researchers to seek out unknown genetic variants, as in rare diseases, or screen for a panel of possible genetic variants all at once. The method has become foundational to oncology, namely in the development of liquid biopsies or companion diagnostics for precision therapies, and it is being investigated for applications in the diagnosis of hereditary hearing and vision loss, genetic cardiomyopathies and autosomal dominant polycystic kidney disease, among others. NGS also permits single cell sequencing, which allows the discovery of biomarkers at the level of an individual cell.
NGS techniques are, at this point, well codified, but a few persistent challenges remain for researchers. One is library preparation. In any NGS screen, researchers start by fragmenting sample DNA into small segments and labelling them for easy identification. The fragments are then sequenced and the results are analysed. That can be a time-consuming process.
“Unfortunately,” Kapustina says, “we cannot skip library prep, but we're trying to make those workflows as compact and convenient as possible.” Thermo Fisher offers a range of standardized reagents that support library preparation for both Ion Torrent and Illumina sequencers, along with expert-led services for those developing specific diagnostics.
Another challenge for researchers is sample stability. “It's very important for people using liquid handlers or robotic systems, as they [add their samples and] leave it for a period of time—up to a few days,” says Sigita Činčiūtė, product manager at Thermo Fisher in Vilnius. Thermo Fisher actively consults with researchers to help them build workflows and diagnostics that will take such considerations into account.
Expanding the diagnostic toolkit
The arrival of technologies suitable for molecular diagnostics is not very common. The molecular diagnostics market is still dominated by decades-old PCR and ELISA tests. The development of CRISPR and NGS diagnostics presents a rare chance to expand that portfolio.
Both techniques have proven easy to adapt and scale, allowing for the rapid development and testing of diagnostic candidates, and they are both relatively low cost, making them more palatable for insurers and health systems. They also complement each other. NGS allows for the identification of unknown variants or the screening for several known biomarkers at once. Single-sequence CRISPR diagnostics are well-suited for use in the field or point-of-care.
It’s unlikely that CRISPR and NGS diagnostics will supplant more established methods. Rather they will almost certainly expand access to different kinds of diagnostic information presently out of reach. Dev, for his part, is already using multiple tools to diagnose prostate cancer.
“Whatever diagnostic tool we end up relying upon, it's going to be multimodal,” he says. “None of these tools are likely to exist in isolation”
That is good news for those pursuing molecular diagnostics, or for the development of CRISPR and NGS applications even further afield in agriculture or biofuels. But most importantly, it is good news for patients.