Long before the COVID-19 pandemic shined a spotlight on CRISPR-based diagnostics, Kiana Aran, PhD, was developing the CRISPR-Chip. It was, according to Aran, the first transistor that uses CRISPR to search the genome for potential mutations. In addition, the graphene field-effect transistor (gFET) did not require amplification or sequencing of the nucleic acid.
Now, two years after its introduction, a group headed by Aran, co-founder and CSO at Cardea Bio, and assistant professor at the Keck Graduate Institute of Applied Life Sciences, is introducing an improved CRISPR-based gFET system, named SNP-Chip.
The two advances in the SNP-chip compared to its predecessor are 1) improved electronics that allow for more, higher quality, measurements and 2) the incorporation of a novel Cas enzyme (a Cas9 ortholog). The ability of the SNP-chip to identify single nucleotide polymorphisms (SNPs) was illustrated in two genetic diseases: amyotrophic lateral sclerosis (ALS) and sickle cell disease (SCD).
The work is published online today in Nature Biomedical Engineering in a paper titled, “Discrimination of single-point mutations in unamplified genomic DNA via Cas9 immobilized on a graphene field-effect transistor.”
This is a nice proof of concept study, asserted Rodolphe Barrangou, PhD, professor in the department of food, bioprocessing, and nutrition sciences at North Carolina State University, and editor-in-chief of The CRISPR Journal, that showcases the potential of the previously developed method, with convincing SCD and ALS targets. “Being able to detect SNPs,” Barrangou continued, “especially without the need for amplification, is noteworthy and opens new avenues for CRISPR-based diagnostics.”
The authors noted that the SNP-Chip is not the first new technology that has been developed to perform SNP genotyping without sequencing. Some technologies in this space have removed the need for expensive optical equipment while others have cut out the need for amplification. But no previous methods have achieved both.
Michael Heltzen, CEO of Cardea Bio, told GEN that he believes the SNP-Chip is, “the biggest genetic breakthrough since PCR was invented in 1983.” While Barrangou asserts that the SNP-Chip “could be a game-changer for precision diagnostics,” he added that it is “not on the same revolutionary scale as PCR.”
Detecting disease-causing SNPs in DNA
For the past two years, Aran told GEN, her team has been working toward point mutation detection in the SNP-Chip. This goal came largely from listening to what their customers wanted.
The current work illustrates the detection of SNPs by the SNP-Chip in two human disease models. When the team tested the SNP-Chip on genomic samples isolated from three patients with SCD and three healthy individuals, the SNP-Chip could differentiate between the two. In ALS, the SNP-Chip could discriminate between genomic DNA extracted from human induced pluripotent stem cells (hiPSCs) from a healthy individual and those from an individual with familial ALS (carrying the H44R mutation in the superoxide dismutase type 1 (SOD1) gene). In the SCD model, the SNP-Chip was also able to detect heterozygosity of SNPs without DNA amplification.
Aran stressed that success in detecting mutations associated with these two diseases is just the beginning. Using a new guide RNA, which would reconfigure the CRISPR-element employed within SNP-Chip, is all that is needed to target different mutations.
A Cas enzyme from a Cas expert
One of Anan’s co-authors is Virginijus Šikšnys, the Lithuanian biochemist who has been in the CRISPR field almost before it existed. Despite his 2012 paper bearing a later publication date than that of the infamous Science paper co-authored by Jennifer Doudna, Emmanuelle Charpentier, and colleagues, his related discoveries were made no later than theirs. (He shared the 2018 Kavli Prize with Doudna and Charpentier.)
Last year, Šikšnys told GEN, with regards to non-genome editing applications, “programmable CRISPR-Cas-based detection technologies will expand because of the ease of changing recognition sequence and their specificity. On this front, we will likely see even more CRISPR-Cas applications in the diagnostics space.” By teaming up with Cardea Bio, Šikšnys is now playing a central role in making this happen.
The first CRISPR-Chip utilized the sequence-specific gene-targeting properties of a deactivated Cas9 enzyme. The deactivated Cas yielded a big signal, according to Aran, but Cardea found the commercial accessibility to be a hurdle. So, Cardea turned to Šikšnys and his company, CasZyme, for alternative Cas orthologs. The Cas9 orthologue that is used in the SNP-Chip comes from Mycoplasma gallisepticum CA06 strain (MgaCas9).
Šikšnys noted that merging a diversity of CRISPR-Cas biology with electronics via Cardea transistors opens up “a whole new range of possibilities for diagnostic and research applications.” He added that “using the Cas9 orthologue for SNP detection is just the tip of the iceberg of opportunities.”
The most immediate application where Aran envisions SNP-Chip making an impact is in monitoring CRISPR-based gene editing quality control processes. The SNP-Chip can ascertain the efficiency of the editing process by measuring how many cells were edited. There will likely be more applications in the SNP-Chip’s future, as it can theoretically detect SNPs in any genetic material—from agricultural and environmental monitoring to human disease.
The CRISPR-based SNP-Chip is dependent on protospacer adjacent motif (PAM), which creates a limitation when targeting a SNP. If a PAM is not near the target site, it will be more difficult to detect because Cas9 cannot gain access. Šikšnys previously told GEN that even the most versatile SpyCas9 variant cannot access approximately 75% of the single-base mutations that are associated with human disease, because there is no appropriately located PAM. Barrangou added that it is important to note “the continued need to encompass orthogonal Cas9 effectors so PAM-related limitations are overcome using biodiversity.”
Could the SNP-Chip detect variants in emerging (and evolving) viruses like SARS-CoV-2? At the moment, one chip is limited to detecting only one point mutation. In order to detect more point mutations, Aran explained, you would need more than one chip. But the team is working on this improvement by creating a 4-plex chip with hopes of a 16-plex chip.
Today, SNP-Chip experiments are done in the San Diego office of Cardea Bio. But the goal, according to Aran, is to sell disposable chips so that anyone can use them, anywhere.
The late Kary Mullis, PhD, inventor of PCR, once said that “science consistently produces a new crop of miraculous truths and dazzling devices every year.” The role that the SNP-Chip will play in the future of CRISPR diagnostics, and other applications, remains to be seen. Nevertheless, it is an early contender in a new era of amplification-free SNP detection.