Date: 8th April 2021
Single-nucleotide polymorphisms (SNPs) account for over 50% of disease-causing mutations in humans and can affect pathogenesis of disease, and how we respond to pathogens, chemicals, drugs and vaccines. The current gold standard for large scale SNP genotyping requires specialised equipment, staff with expertise, and involves lengthy processes, rendering these technologies incompatible at the point of care or in the field. Now, researchers have developed a new biosensor for the electronic detection of unamplified target genes using CRISPR-powered transistors, dubbed ‘SNP-Chip’, which can discriminate SNPs within an hour.
One of the most exciting emerging technologies of 2019 was the CRISPR-Chip. Developed by Kiana Aran, at the Keck Graduate Institute, US, it was the first transistors that combined CRISPR technology to search the genome for potential mutations, with the use of ultra-sensitive graphene. The biosensor was constructed by utilising the gene-targeting ability of catalytically deactivated Cas9 (dCas9) protein complexed with a specific targeting single-guide RNA, which was then immobilised on the surface of graphene-based transistors. The biosensor would scan the genome for its target DNA sequence and, when bound, the altered conductive properties of the graphene were subsequently detected with a simple handheld reader.
Then, 6 months later came the announcement of a proposed business combination to develop CRISPR-Chip which combined the technology from two innovative companies based in San Diego, US, both of which are led/ co-founded by Kiana Aran and Michael Heltzen. Nanosens Innovations provided the CRISPR-Cas9 nucleotide detecting technology, whilst Cardea Bio used their revolutionary graphene biosensor platform, and Nanosens became a subsidiary of Cardea. The resulting Genome Sensor™ was likened to googling the genome, unlocking our DNA and opening up endless possibilities.
Now, Aran and colleagues report a new improved CRISPR-based graphene field-effect transistor (gFET), referred to as SNP-Chip, which is capable of detecting single-nucleotide mutations in a target DNA sequence without the need for amplification. The work is a result of a collaborative effort between Cardea Bio, the Keck Graduate Institute (KGI), University of California Berkeley, University of California Irvine, Vilniaus University, and CasZyme.
In the paper published in Nature Biomedical Engineering journal this week, the team explored the activity of 3 different Cas9 variants, dCas9 used in the original CRISPR-Chip, SpCas9, and MgaCas9 to improve SNP discrimination, and expanded the types of electrical measurements taken to ensure high quality readings which allowed them to achieve single-nucleotide specificity.
One key limitation to the technology is the PAM-dependence nature of CRISPR-Cas9 to interact and bind to its target DNA however, with the growing discovery of Cas9 orthologues that have different PAM requirements this constraint may be overcome. Here, the team tested the nuclease-active Cas9 from Streptococcus pyogenes, which is the most widely used CRISPR enzyme, and Mycoplasma gallisepticum CA06 strain (MgaCas9), a more recently discovered orthologue that uses a different PAM sequence to recognise potential targets.
The SNP-Chip was designed to detect SNPs in target genes in two human disease models, sickle cell disease (SCD) associated with a point mutation in the HBB gene, which encodes the globin protein – haemoglobin subunit beta, and amyotrophic lateral sclerosis (ALS), a neurodegenerative disease which is associated with a SNP in the superoxide dismutase type 1 (SOD1) gene.
The SNP-Chip utilising either dCas9 or Cas9 could discriminate between genomic DNA samples from healthy patients and those with SCD rapidly and accurately. However, it could not discriminate between heterozygous and homozygous samples. In contrast, a SNP-Chip functionalised with MgaCas9 was able to target the SCD-associated SNP within the PAM and therefore was able to directly detect the differences between these patient samples. In the second disease model, the SNP-Chip could also differentiate between genomic DNA carrying the SOD1 SNP and wild type samples.
The team have presented a new version of a gFET sensor, SNP-Chip, that can detect point mutations, in unamplified genomic DNA, in less than an hour, demonstrating the platform’s potential to streamline genetic testing for research and diagnostic purposes. With the ongoing discovery of new Cas9 variants the natural limitations of technology regarding PAM-related sequence restrictions can be overcome by exploiting biodiversity.
The demonstration of the highly sensitive and quantitative ability of SNP-Chip to detect heterozygosity will be an important one. As such this technology will be able to quantify predisposition to many genetic diseases and will have implications for those that are inherited.
The technology is highly programmable and can be easily reconfigured by using guide RNAs to target virtually any genetic disease, and together with increasing availability of Cas protein the platform offers the scope to revolutionise the screening for genetic mutations.
However, the applications will extend far beyond diagnostics, the SNP-Chip has the potential to provide valuable and actionable insight into areas like agriculture, industrial bioprocesses, and even evolutionary change – where mutations confer resistance to antibiotics or mutating viruses.
CRISPR technologies are driving a new generation of therapeutics and gene therapies such as we have recently seen with in vivo editing of bone marrow cells for the treatment of SCD, or treating muscular dystrophy, in preclinical trials. In addition, several clinical trials have also commenced – in the main using gene edited cells to treat diseases such as refractory cancer, multiplexed CRISPR-Cas9 gene edited cells have also been used which adds additional layers of complexity. In a world’s first – last year the first human was treated with in vivo CRISPR editing therapy. Here the biosensor offers an immediate platform to monitor such CRISPR-based gene edits, and in light of the increasing numbers and diverse range of such therapies the SNP-Chip will offer an invaluable and powerful tool to ascertain the efficiency of CRISP-edits, and to increase the quality control of such therapeutics.
Currently, one limitation is the one-chip one-mutation detection ability of the biosensor. Improvements on this front are aimed at creating multiplexed chips able to detect multiple SNPs. The team also hope to manufacture disposable chips that will facilitate in-field applications together with their handheld portable reader. The diversity of CRISPR-Cas biology married with electronic detection technologies will open a whole new avenue of possibilities for diagnostic and research applications.
For more information please see the press release from Cardea Bio at Business Wire
Balderston, S., J. J. Taulbee, E. Celaya, K. Fung, A. Jiao, K. Smith, R. Hajian, G. Gasiunas, S. Kutanovas, D. Kim, J. Parkinson, K. Dickerson, J.-J. Ripoll, R. Peytavi, H.-W. Lu, F. Barron, B. R. Goldsmith, P. G. Collins, I. M. Conboy, V. Siksnys and K. Aran (2021). “Discrimination of single-point mutations in unamplified genomic DNA via Cas9 immobilized on a graphene field-effect transistor.” Nature Biomedical Engineering.