ToloBio Secures Key U.S. Patent for CRISPR-Based Rapid Diagnostic Technology, Files IPO Prospectus

On a day in late February, news from the United States Patent and Trademark Office brought joy to Dr. Wang Jin in Shanghai. After months of communication and responses to office actions, the Cas12 protein diagnostic patent filed by ToloBio, which he co-founded, through the Patent Cooperation Treaty (PCT) in the United States was finally granted. This represents a significant milestone in ToloBio’s global patent strategy.

Dr. Wang Jin told VCBeat that Tulugang Biotechnology would further consolidate its leading patent position in the field of CRISPR diagnostics by collaborating with its U.S. partner, Team Sherlock Bio. With this development, Chinese biotech companies have won a key battle in the long-standing CRISPR patent dispute for the first time.

Unlike in traditional biotechnology, where domestic players have largely been limited to catching up and imitating, Chinese innovative enterprises developing CRISPR technology start from a higher baseline and will undoubtedly face many novel questions for which there are no reference answers. One of the most thorny new issues is the patent disputes that are likely to accompany the entire cycle from technological innovation to product innovation.

Dr. Qi Fei, Executive Director at Legend Capital, reflected emotionally on this patent dispute: “This may be the most significant moment for China’s gene editing sector. In the life sciences field, it has been uncommon to witness competition over such foundational patents, let alone in the realm of revolutionary technologies like gene editing. The fact that Chinese scientists and enterprises have achieved genuine global original innovation and defeated all competitors in securing patents marks a milestone for foundational patents in China’s life sciences sector. Henceforth, Chinese scientists and enterprises have secured a place in the landscape of global foundational patents in gene editing.”

Compared with the Cas12 protein, the Cas9 protein used in CRISPR/Cas9 gene editing is more widely recognized; it is the key enzyme underpinning CRISPR-based gene editing.

Let’s briefly expand on some background knowledge. The full name of the CRISPR/Cas system is “Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein system.” It is an adaptive immune system found in most bacteria and the vast majority of archaea, primarily functioning to eliminate foreign plasmids or bacteriophages. To date, three different types of CRISPR/Cas systems have been identified, covering approximately 40% of sequenced bacteria and 90% of sequenced archaea. Among these, the type commonly used in gene editing features a relatively simple composition, centered on the Cas9 protein and guide RNA (gRNA).

As an unprecedented, more efficient, and cost-effective gene-editing technology, the CRISPR/Cas system has completed proof-of-concept and early-stage product development within just a few years of its emergence, demonstrating strong commercial potential in clinical settings and becoming one of the hottest biotechnologies.

Specifically, the initiative for the commercialization of CRISPR/Cas systems has been primarily held by its discoverers and patent holders. In 2012, scientists Jennifer Doudna and Emmanuelle Charpentier elucidated the editing mechanism of the CRISPR/Cas9 system. Eight years later, they were awarded the Nobel Prize in Chemistry for this breakthrough. Meanwhile, the two scientists swiftly transformed this scientific discovery into an innovative technology that revolutionized gene editing. To this end, Charpentier founded CRISPR Therapeutics, while Doudna established Intellia Therapeutics, both focusing on the development of gene-editing therapies based on CRISPR/Cas9 technology. In 2016, these two companies, armed with novel biotechnologies, went public and garnered strong interest from global investors.

On the other hand, Feng Zhang, a Chinese-American scientist at the Broad Institute of MIT and Harvard, secured multiple key core patents for CRISPR-Cas9 by pioneering the application of gene-editing technology in human cell therapy.

In 2013, armed with patented inventions, Zhang Feng founded Editas Medicine to explore the use of CRISPR/Cas9 technology for treating various diseases. From its inception, the company was highly sought after by top global venture capital firms. In its Series A and Series B financing rounds, Editas Medicine secured $43 million and $120 million, respectively, with investors including Google Ventures. Also in 2016, Editas Medicine went public on the NASDAQ stock exchange in the United States.

Editas Medicine focuses on innovative therapies for cancer and genetic disorders based on CRISPR/Cas9 technology, having developed multiple programs. These include a version 2.0 CAR-T cell therapy co-developed with JUNO Therapeutics, clinical studies targeting Leber congenital amaurosis, and gene-editing therapies for genetic diseases such as sickle cell anemia and herpes keratitis. By March 2020, Editas Medicine’s CRISPR-based therapy AGN-151587 (EDIT-101) had completed patient dosing in its Phase I/II clinical trial for the treatment of Leber congenital amaurosis type 10 (LCA10), marking the world’s first in vivo administration of CRISPR gene editing. The company appears to be just one step away from translating cutting-edge technology into blockbuster drugs.

In a sense, once the operational principles of the CRISPR system were mastered, the small Cas protein acquired the ability to turn stone into gold.It is precisely for this reason that the protracted and fierce battle over foundational patents in the field of gene-editing therapies has persisted for a decade since the technology’s inception, becoming a focal point for the global scientific community. In February 2022, the decade-long rivalry between Feng Zhang and Jennifer Doudna concluded with a preliminary victory for Zhang. However, disputes over foundational patents covering additional Cas proteins and technological applications remain unresolved.

As proteins such as Cas13 and Cas12 were successively discovered, they quickly attracted widespread attention. With the continuous influx of technology and funding, CRISPR-based rapid diagnostic methods leveraging these proteins have been rapidly deployed in applications such as tumor detection and pathogen screening. Driven by the demand for nucleic acid testing during the COVID-19 pandemic, these technologies have stepped into the global spotlight.

In 2022, Nature published its annual list of technologies to watch, with CRISPR-based diagnostics featured among them. Research has shown that by leveraging and exploring the characteristics of Cas proteins such as Cas12 and Cas13, CRISPR diagnostics can detect a broad range of pathogens and even effectively diagnose other non-infectious diseases. Since 2018, the number of research publications on CRISPR detection technologies has steadily increased, exceeding one thousand by 2021. Correspondingly, CRISPR detection technologies have become increasingly mature and continue to expand in scope. At present, the relatively mature CRISPR-based rapid diagnostic systems developed worldwide includeSHERLOCK diagnostic system, HOLMES diagnostic system, DETECTR diagnostic system, etc.。

In fact, when it comes to the rapid diagnostic applications of CRISPR/Cas systems, Cas9 protein remains the first choice that comes to mind. In 2016, Collins et al. developed a rapid and cost-effective method for detecting the Zika virus based on the ability of CRISPR/Cas9 to specifically recognize and cleave target sequences. By combining Cas9 protein with PCR technology, they used sgRNA to specifically recognize PAM sequences present only in the genome of the American strain of the Zika virus, enabling precise differentiation between subtypes within three hours. However, due to the lengthy processing time and the need for temperature-variable reactions, this system has not been widely adopted in the development of diagnostic products.

Subsequently, researchers began exploring the use of Cas13 proteins for CRISPR-based diagnostics. The Cas13 protein was first discovered in 2015. At that time, the laboratory of Eugene Koonin, an evolutionary biologist at the National Center for Biotechnology Information (NCBI), collaborated with Feng Zhang’s team. Using computational biology approaches, they were the first to identify CRISPR-Cas13 systems capable of RNA editing within microbial metagenomic databases. These systems include the Cas13a (C2c2), Cas13b (C2c6), and Cas13c (C2c7) proteins. They subsequently demonstrated that these systems could efficiently knock down target RNA in mammalian cells. However, when attempting to develop RNA-editing therapeutics, they found that the relatively large size of Cas13a/b/c/d proteins made it difficult to package them into a single adeno-associated virus (AAV) vector for in vivo delivery.

Until 2017, Feng Zhang’s team leveraged the “collateral activity” of CRISPR-Cas13a to establish a rapid nucleic acid detection method. The so-called “collateral activity” refers to the phenomenon whereby, upon binding to a specific target RNA, the Cas13a protein is activated to randomly cleave other non-target RNAs. When combined with isothermal amplification techniques (such as RPA), this enables rapid detection of target RNA. In a series of studies, Feng Zhang’s team applied this detection method to identify specific strains of Zika and dengue viruses, differentiate pathogenic bacteria and human genotypic DNA, and detect mutations in cell-free tumor DNA, naming the platform SHERLOCK.

Similar to the CRISPR/Cas9 system, the CRISPR-Cas13a system was rapidly applied to product development shortly after its discovery. In March 2019, Sherlock Biosciences, founded by Feng Zhang and his team, secured $35 million in funding shortly after its establishment. Licensed through the Broad Institute, the company leveraged CRISPR technology for rapid diagnostics, primarily developing point-of-care testing products for pathogen detection, genotyping, drug-resistance genes, and liquid biopsy for tumors. In May 2020, Sherlock Biosciences’ COVID-19 test received FDA Emergency Use Authorization (EUA), becoming the world’s first registered and approved CRISPR-based diagnostic reagent.

Meanwhile, the CRISPR-Cas12 system has also come into view. The team led by Feng Zhang was also at the forefront of the initial discovery of the Cas12 protein. In 2015, they discovered that this novel CRISPR-Cas protein possesses RNA-guided specific DNA endonuclease activity similar to the commonly used Cas9 protein. Furthermore, it requires only crRNA to guide the specific cleavage of double-stranded DNA, generating sticky ends, among other features, making it suitable for CRISPR-based gene editing.

Subsequently, Dr. Wang Jin’s team and Jennifer Doudna’s team almost simultaneously discovered that the Cas12 protein possesses a novel cleavage activity targeting single-stranded DNA (ssDNA). Guided by crRNA, upon recognizing and binding to the target DNA, Cas12 is activated to exhibit indiscriminate cleavage activity against ssDNA, a phenomenon known as trans-cleavage activity.

Interestingly, in 2017, Jennifer Doudna, the Nobel laureate mentioned earlier for her outstanding contributions to CRISPR gene editing, founded Mammoth Biosciences, once again competing with companies established by Feng Zhang. This company holds a license from the University of California for technology related to the trans-cleavage activity of CRISPR-Cas12 and is exploring the use of CRISPR technology for rapid diagnostics in infectious diseases, oncology, and genetic mutations. The CRISPR-based rapid diagnostic method employed by Mammoth Biosciences is known as the DETECTR diagnostic system. Developed by Jennifer Doudna’s team in 2018, this method is based on the Cas12a protein—the same protein at the center of the patent dispute between Mammoth Biosciences and the Chinese innovative enterprise Tuluogang Biotech, as mentioned at the beginning of this article.

In China, Dr. Wang Jin and Tolo Biotech similarly leveraged the trans-cleavage activity of Cas12a to develop the HOLMES diagnostic system, which predates the DETECTR system. Building on this foundation, Dr. Wang Jin’s team repeatedly validated that both ssDNA and dsDNA targets can activate the trans-cleavage activity of Cas12a, demonstrating the universality of this activity. This work provided a crucial theoretical basis for the subsequent screening and evolution of Cas12a proteins within the HOLMES platform.

According to Dr. Wang Jin, to simplify the operational workflow of CRISPR-based rapid diagnostics and improve accuracy, Tulougang Biotechnology has developed the HOLMESv2 system based on LAMP-Cas12b, establishing a one-step CRISPR diagnostic platform. This is the world’s first one-step CRISPR diagnostic system to be developed, and multiple research teams are currently following up with related studies.

At the end of 2020, Mammoth Biosciences’ COVID-19 diagnostic test based on the DETECTR platform also received Emergency Use Authorization (EUA). Furthermore, to address the need for large-scale screening, the FDA granted EUA in early 2022 to another product under the DETECTR platform, the DETECTR BOOST SARS-CoV-2 assay. Also based on the CRISPR system, this assay integrates CRISPR technology with automation, enabling high-throughput detection via CRISPR for the first time and playing a significant role in mass screening during the global pandemic.

“CRISPR-based diagnostic technologies are rapidly transitioning from the laboratory to clinical applications in China.” Dr. Wang Jin told VCBeat that Chinese companies have kept pace with global advancements in establishing CRISPR diagnostic standards, building supply chain ecosystems, commercializing technologies, and developing product strategies. During the COVID-19 pandemic, Shanghai Bojie also leveraged CRISPR diagnostic technologies licensed from Tolo Biotech to develop rapid COVID-19 test kits, successfully obtaining product registration certificates from the National Medical Products Administration (NMPA). Furthermore, Dr. Wang Jin revealed that Tolo Biotech has established product or technical collaborations with multiple companies in the clinical and environmental monitoring sectors, jointly fostering a thriving ecosystem for this innovative technology.

The emergence of CRISPR-based diagnostic technologies has brought new hope to the long-quiet field of precision medicine. According to foreign media reports, in recent years, the two aforementioned U.S. companies developing rapid diagnostic technologies based on the CRISPR-Cas platform have collectively raised over $500 million in financing. This has attracted investment from industry-savvy firms such as Illumina Ventures, Baidu Venture Capital (BV), Pacific 8 Ventures, and Foresite Capital, laying the foundation for subsequent product commercialization and large-scale application. In China, Tulugang Biotechnology has also secured investments from leading institutions including BGI Win-Win, Legend Capital, and Huiyuan Capital.

According to incomplete statistics from VCBeat, the underlying technology patents for CRISPR diagnostics worldwide are currently held primarily by the three aforementioned companies. The ownership of diagnostic patents for Cas13 is relatively clear, whereas that for Cas12 remains highly uncertain. Specifically, Sherlock Biosciences has obtained licensed Cas13 diagnostic patents from Dr. Feng Zhang’s team at the Broad Institute. In contrast, both Tuluogang Biotech and Mammoth Biosciences claim to hold the underlying Cas12 diagnostic patents, derived respectively from Dr. Jin Wang’s team and Professor Jennifer Doudna’s team. As the two companies’ Cas12-based technical approaches are nearly identical, they are destined to face inevitable competition in their future commercialization efforts.

The patent dispute marks the first confrontation between rivals Tuluogang Biotech and Mammoth Biosciences. VCBeat has previously published an analysis noting that both Tuluogang Biotech and Mammoth Biosciences, whose technologies are highly similar, are expanding into the global market and filing technical patents in multiple countries worldwide. The core of their dispute centers on U.S. patents. Previously, the United States Patent and Trademark Office (USPTO) granted the patent for Cas12-based diagnostics to Mammoth Biosciences. However, Tuluogang Biotech secured its U.S. patent through the Patent Cooperation Treaty (PCT) route, leveraging a priority date more than four months earlier than that of its competitor. Despite this clear advantage in filing priority, Dr. Wang Jin and Tuluogang Biotech have been cautiously preparing and carefully navigating the complex global patent landscape, aiming to secure the most critical foundational patents for CRISPR diagnostic technology.

“We anticipated early on that competition in this field would be fierce, particularly regarding core patents. We made corresponding preparations, but still fell short in starting early enough, doing enough, and going deep enough,” Dr. Wang Jin once lamented to VCBeat. At that time, the U.S. patent dispute between Tuluogang Biotech and Mammoth Biosciences remained unresolved.

Now that the U.S. patent for Cas12 proteins has finally been settled, it marks a victory for the team of Chinese scientists represented by Dr. Wang Jin as global innovation pioneers in the field of cutting-edge biotechnology. It also serves as an example of Tulugang Biotech, a Chinese innovative biotechnology company, competing on the global stage. Even when facing the strongest competitors and the most sought-after technologies, Chinese scientists and enterprises can achieve critical victories through genuine original innovation and persistent efforts.

Prior to this, Tuluogang Bio and Sherlock Biosciences had already signed a cooperation agreement. Building on their existing collaboration in the Greater China and U.S. markets, the two parties further executed a global “CRISPR Diagnostics Patent Cross-License” cooperation agreement.

As the only two companies in the market holding foundational patents for CRISPR-Cas12 and Cas13 molecular diagnostics, this agreement further strengthens the in-depth collaboration between Tuluogang Biotech and Sherlock Biosciences, providing both parties with the most comprehensive portfolio of rights to CRISPR diagnostic-related patents. This ensures that both companies can launch more competitive CRISPR diagnostic products in the global market.