Mammoth Biosciences: Going Beyond Genetic Literacy
It was your typical rainy Vancouver Saturday morning, I was reading my copy of Walter Isaacson’s “The Code Breaker” and I was on page 421. I had just made my way through an exciting rendition of Jennifer Doudna’s experience kick-starting the Berkley’s Lab coronavirus centre when the chapter 51 heading caught my attention: Mammoth and Sherlock.
The chapter continued on with documenting Zheng and Doudna’s competition, but also emphasized the discovery of the DETECTR (DNA endonuclease targeted CRISPR trans reporter). The DETECTR a diagnostic tool that kick-started Mammoth Biosciences, and is a combination of the Cas12a enzyme and CRISPR.
The Cas12a enzyme has a very special property: it can be targeted like Cas9 to find and cut a specified sequence of DNA, but it can also afterwards start cutting and chopping up any single-stranded DNA nearby. Knowing this, the co-founders added a fluorescent signal connected to a bit of DNA to serve as a “reporter” molecule to a CRISPR-Cas12 system. The result of this combination was a diagnostic tool that could detect whether the patient had a particular virus, bacteria or cancer.
The doctoral students and Doudna applied the DETECTR system to the human papillomavirus (HPV), put up an article detailing their research in February of 2018, and launched Mammoth Biosciences two months later.
Before we dive into the science, it is key to quickly go over the genetic text within the HPV virus, and how Mammoth Biosciences addressed that.
HPV: Human Papillomavirus
Human Papillomavirus (HPV) has been labelled the most commonly sexually transmitted agent worldwide, both in its clinical and subclinical presentations in men and women. The key facet of HPV is its relationship with cervical cancer in women (80% of cases in most developing countries, with an annual incidence of almost half a million and a mortality rate of approximately 50%).
About 189 HPV genotypes have been sequenced and classified according to their biological niche and oncogenic potential. Across the range of HPV-related cervical lesions, HPV-16 is the most frequent HPV type. The most prevalent HPV types in women with ICC (invasive cervical cancer) are HPV-16,18,33,45,31, and 58, but certain genotypes have been associated with varying clinical outcomes.
HPV has co-existed within its human host for a very long time and has evolved into different evolutionary lineages, and it is now well-recognized thanks to modern-day synthetic biology.
Within an icosahedral capsid (geometric box with 20 sides containing the virus), the HPV virion possesses a double-stranded, circular DNA genome of roughly 7900bp, with eight overlapping open reading frames, including (E) early (in yellow on the left) and (L) late genes (in red on the left), as well as an untranslated lengthy regulatory region (in blue on the left).
The major and minor capsid proteins are encoded by the L1 and L2 genes.
The early genes control viral replication, and some of them have the ability to change cells. L6 and L7 are late genes that code for structural capsid proteins that encase the viral DNA.
Papillomavirus infection involves virus particles gaining entrance to the epithelium basal layer and dividing basal cells. The HPV virus enters the nucleus of a basal epithelial cell after entering epithelial tissues, where early genes E1 and E2 are produced, replicating the viral genome and producing messenger RNA required for viral replication.
The tools at Mammoth Biosciences today are looking at reading genetic codes in order to diagnose HPV on the molecular level. Think a quantity multiplied by 6.02x10²³!
DETECTR: What is it exactly?
So, what is the science behind the DETECTR tool?
Note: to provide an accurate overview of the DETECTR product, I am using the company’s initial research paper, found here.
Essentially, Cas12a is targeted to a specific DNA sequence, such as the Human papillomavirus (HPV) genome, through a crRNA (also known as CRISPR RNA a 17–20 nucleotide sequence complementary to the target DNA). Then, an ssDNA-fluorescently quenched (FQ) reporter, which will produce a signal when the ssDNA is degraded, is added to the reaction.
The DNA is initially amplified by RPA (replication protein A is the major protein that binds to single-stranded DNA (ssDNA) in eukaryotic cells) using isothermal amplification to improve sensitivity.
Cas12a’s DNase activity is triggered when Cas12a-cRNA base pairs with the dsDNA of interest. Afterwards, Trans-ssDNA in the area, including the ssDNA-FQ reporter, is then degraded at a rate of 1,250 cuts per second. The presence of the DNA of interest, in this case, HPV, is shown by a measurable fluorescence signal.
The Mammoth lab proved that DETECTR could accurately discriminate between two forms of HPV, HPV16 and HPV18, from human cells at attomolar levels in less than an hour as proof of concept.
Therefore, DETECTR has the ability to rapidly detect nucleic acids with high selectivity from patient samples.
The Mammoth Bioscience Platform
Mammoth Biosciences, a recent unicorn with a valuation of $1 billion, focuses its platform on one key player: enzymes. Their CRISPR has the largest collection of Cas enzymes, driven by its proprietary protein discovery engine. The unique properties of these enzymes such as their small size, temperature stability, faster reaction kinetics, high-fidelity and expanded targeting capabilities have the potential to revolutionize the CRISPR diagnostic field.
Diagnostics is truly the area in which Mammoth Biosciences could distinguish themselves with, and currently, they are doing so with key partners. For instance, The Defense Advanced Research Projects Agency (DARPA) for a pathogen detecting test that should detect up to 10 pathogens at once.
This company has also partnered with the National Health Institute and their Rapid Acceleration of Diagnostics (RADx) program to work on developing better diagnostic tools (like DETECTR) using CRISPR.
By continuing to work on Cas12, Cas13, Cas14, and Casɸ, Mammoth Biosciences can provide enhanced diagnostics and gene editing tools for a vast area of fields like life science research, agriculture and much, much more.
Mammoth Biosciences, a company within the ranks of unicorns, is harnessing the diversity of the book within humans to create the next generation of CRISPR products. With a team consisting of prize-winning PhDs and Jennifer Doudna, a 2020 Nobel Laureate for her discovery of CRISPR, Mammoth Biosciences is on its way to increasing humanity's genetic literacy by allowing us to read and write the code of life.