CasX Enzymes: A New Family of RNA-Guided Genome Editors

  • Search of Unusual Microbes Yields New CRISPR-Cas Systems
  • Tiny Life Forms Have Smallest Working CRISPR-Cas Systems
  • Novel CasX Structure and Mechanism Characterized by Cryo-Electron Microscopy

In 2012, a Science magazine publication by Doudna, Charpentier, and coworkers describedCas9, the CRISPR-associated (Cas) protein, as a programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. This work has already been cited ~6,500 times, alongside two other Cas9 studies listed in PubMed that year. They have been followed by a steadily increasing number of annual Cas9-related publications, as show in the chart below. A large part of this growing interest is due to the proven utility of CRISPR-Cas9, and variants thereof, for gene editing, which I have previously blogged about.

Given the broad scientific, clinical, and commercial utility of CRISPR-Cas systems, it is not surprising that there has been considerable effort directed toward either engineering analogs of known Cas enzymes, or discovering new homologs in unexplored organisms. With regards to the latter approach, Doudna, Banfield and coworkers noted in 2017 that the then available CRISPR-Cas technologies were based solely on systems from isolated, cultured bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped.

They added that metagenomics—sequencing DNA extracted directly from natural microbial communities—provides access to the genetic material of a huge array of uncultured organisms. For this reason, through use of metagenomics, the researchers were able to discover two previously unknown CRISPR-Cas systems. These new Cas proteins, named CasX and CasY to designate as yet unknown specifics, are said to be among the most compact systems yet discovered. In February 2019, as a follow-up to these discoveries, Doudna and collaborators published the mechanistic details for CRISPR-CasX in Nature magazine. This will be the focus of this blog, but before that story, here are some introductory comments about metagenomics, a transformative technology in its own right.

Putting Together All of the Pieces

In a review by Chen and Pachter, metagenomics is described as “the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species.” They add that “metagenomics has revolutionized microbiology by shifting focus away from clonal isolates towards the estimated 99% of microbial species that cannot currently be cultivated.”

A typical metagenomics project begins with the construction of a DNA library derived from a minimally processed environmental sample that is usually comprised of multiple different genomes with different copy numbers. The increasing capacity of factory-like sequencing centers has facilitated whole-genome shotgun sequencing and genome assembly of these complex mixtures. At the risk of oversimplification, this to me is conceptually akin to simultaneously putting together correctly all of the pieces of multiple different jigsaw puzzles.

There are many technical variations for these sequencing and bioinformatic procedures, but at a high-level, these can be categorized as either using extracted DNA per se (metagenomics) or cDNA derived from reverse transcription of extracted RNA (metatranscriptomics). Both of these approaches were used in the aforementioned discovery of CasX and CasY, starting with quite unusual sample sources: (1) acid-mine drainage samples (from the Richmond Mine at Iron Mountain in California); (2) river water and sediment samples (from a site along the Colorado River in Colorado); and (3) cold, CO2-driven geyser water (from Crystal Geyser on the Colorado Plateau in Utah pictured here). Presumably, these relatively unusual sample sources increased the discovery-probability, as scientists were able to examine the previously unknown organisms present in each sample.

Discovery of CasX and CasY

Using metagenomics, Doudna, Banfield and coworkers found a number of CRISPR-Cas systems, including what they believed to be the first Cas9 in the in the archaeal domain of life. Archaea constitute a domain of single-celled microorganisms. These microbes are prokaryotes, meaning they have no cell nucleus. Archaeal cells have unique properties that separate them from the other two domains of life, bacteria and eukarya, as depicted here. Archaea are further divided into multiple recognized phyla, but classification is difficult, as most have not been isolated in the laboratory.

This divergent Cas9 protein was found in little studied nanoarchaea, as part of an active CRISPR-Cas system. Incidentally, nanoarchaea are “nano” indeed, only ~400 nm in diameter—about 5% of the volume of your archetypical 1 μm3prokaryote, according to one estimate—andNanoarchaeum equitansharbors a genome that is only 480 kb. Also discovered were two previously unknown Cas proteins unlike all the previous Cas proteins. These were named CasX and CasY, since it was not clear what they actually did. CasX and CasY are among the most compact systems yet discovered, according to these researchers, who concluded that “interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.”

Cryo-Electron Microscopy (cryo-EM) Characterization of CasX

In February 2019, a follow-up report in Natureby Doudna and collaborators focused on the mechanistic details for CRISPR-CasX. Although RNA-guided DNA binding and cutting proteins have proven to be transformative tools for genome editing across a wide range of cell types and organisms, only two kinds of CRISPR-Cas nucleases—Cas9 (depicted here) and Cas12a (aka Cpf1)—provide the foundation for this revolutionary technology.

The only conserved part of CasX, the RuvC domain, shares less than 16% identity with RuvC domains in either Cas9 or Cas12a. This evolutionary ambiguity in CasX hinted that this enzyme may have a structure and molecular mechanism distinct from that of other CRISPR-Cas enzymes. These structural and mechanistic questions were investigated by use of cryo-EM, a specialized method recently catapulted into widespread view by the co-awarding of the 2017 Nobel Prize in Chemistry to its three pioneers.

As discussed in an introductory YouTube video on cryo-EM, scientists traditionally used X-ray crystallography to obtain biomolecular structures, which requires growing suitable crystals that are oftentimes extremely difficult or not possible to obtain. However, as seen here, freezing a thin layer of a solution of the sample for cryo-EM enables the technique to handle structures for which crystallography is not a viable option. In addition, cryo-EM can visualize much larger structures than crystallography can—100-fold larger according to one cryo-EM expert. By way of example, a 1.8-Å-resolution structure of 334-kDa glutamate dehydrogenase, and 3.6-Å-resolution structure for 11,200-kDa Dengue virus have been reported.

Scientist preparing samples for cryo-EM under liquid nitrogen temperature

Doudna and collaborators took advantage of cryo-EM to obtain eight molecular structures of CasX in different states, which interested readers can view by consulting the 2019 Nature publication (unfortunately, copyright restrictions prevent reproduction here). The researchers’ verbal description of what was found highlights the following structural elements:

“An unanticipated quaternary structure in which the RNA scaffold dominates the architecture and organization of the enzyme. Phylogenetic, biochemical and structural data show that CasX contains domains distinct from—but analogous to—those found in Cas9 and Cas12a, as well as novel RNA and protein folds; thus establishing the CasX enzyme family as the third CRISPR-Cas platform that is effective for genetic manipulation. Finally, distinct conformational states observed for CasX suggest an ordered non-target- and target-strand cleavage mechanism that may explain how CRISPR–Cas enzymes with a single active site, such as Cas12a, achieve double-stranded DNA (dsDNA) cleavage. The small size of CasX (<1,000 amino acids), its DNA cleavage characteristics, and its derivation from non-pathogenic microorganisms offer important advantages over other CRISPR–Cas genome-editing enzymes.”


On the basis of their functional and structural data, Doudna and collaborators propose a model of CasX activation and DNA cleavage that includes the following steps: (1) guide RNA binding-induced CasX structural stabilization and DNA search; (2) non-target-strand binding-assisted DNA unwinding, R-loop formation and nontarget-strand loading into the RuvC active site; (3) RNA-DNA hybrid duplex bending with the aid of the proposed target-strand loading (TSL) domain to position the target DNA strand for cleavage; and (4) product release after the cleavage of both DNA strands.

They added that two distinct target DNA-bound states indicate that CasX coordinates sequential dsDNA cleavage by its single RuvC nuclease, using the zinc-finger-containing TSL domain. Also, the TSL domain appears to confer a convergent mechanism of acute target-strand DNA bending that is central to all type V single-nuclease CRISPR-Cas enzymes.

Looking forward, they speculated that “[t]he compact size, dominant RNA content and minimal trans-cleavage activity of CasX differentiate this enzyme family from Cas9 and Cas12a, and provide opportunities for therapeutic delivery and safety that may offer important advantages relative to existing genome-editing technologies.”

In my opinion, it will likely take some time and considerable experimentation by the scientific community to assess whether any of these potential advantages offered by CasX will actually pan out and lead to widespread adoption. In the meantime, mRNA-encoding Cas9 has firmly established its utility and enjoys extensive adoption, as exemplified by many diverse applications that I found among the search results for “TriLink AND Cas9” in Google Scholar.

As usual, your comments are welcomed.

Highlights from the 14th Annual Meeting of the Oligonucleotide Therapeutics Society (OTS 2018)

  • Nearly 600 Attendees from Around the Globe Converge in Seattle
  • Four Full Days of Nucleic Acid-Based Therapeutics Talks and Posters by Academics and Companies
  • Jerry Comments on His OTS 2018 Favorites

The idea for the OTS was conceived in 2001 by an international group of renowned oligonucleotide scientists, who hoped to bring together expertise from different angles of oligonucleotide research and create synergies to help the field accomplish its full therapeutic potential. The vision included a new era of oligonucleotide drugs that would change the landscape of therapeutic modalities.

This prescient view of a “new era of oligonucleotide drugs” has indeed become reality, and has expanded to include much longer polynucleotides, namely mRNA. The OTS 2018 meeting held on September 30 – October 3 in Seattle, WA, attracted nearly 600 participants from around the world, pictured in this group photo. There were many excellent talks and posters, and before commenting on my favorites among them, I have attempted to distill into a few words my overall impressions about what was “hot,” “new”, and “different” at OTS 2018 compared to past meetings.

  • Credit: Prof. Steven Dowdy, Event Chair

    CRISPR is definitely “hot,” based on the large number of CRISPR talks and posters, which complemented CRISPR “rock star” Dr. Feng Zhang’s exciting Keynote Presentation titled Genome Editing Technologies and Beyond. Here are links to his 83 CRISPR-related publications, and my previous blogs related to CRISPR.

  • What struck me as “new” are the advances in discovery and development of innovative compositions for improved and/or more specific delivery of oligonucleotide and siRNA therapeutics, as well as longer mRNA as drugs. In the next section, I’ll provide an example of research that deals with both of these “new” and “hot” topics.
  • As for what’s “different,” I fully agree with session chairperson Laura Sepp-Lorenzino (Vertex Pharmaceuticals, Inc.), who openly stated that previous meetings, especially early ones, were peppered with “challenges and issues.” Now, there is a preponderance of positive preclinical results and—most importantly—FDA approval of oligonucleotide drugs, along with a promising pipeline.

OTS Young Investigator Award

Prof. Yizhou Dong. With his permission.

This year’s Young Investigator Award was presented to Prof. Yizhou Dong, an Associate Professor at the Ohio State University College of Pharmacy. His presentation titled Development of Nanomaterials for mRNA Therapeutics and Genome Editing described the development of novel nanoparticles for delivery of Cas9 mRNA for improved CRISPR editing of genomic DNA. This exciting talk encompassed both the “new” and “hot” topics at OTS 2018 that I mentioned above.

Prof. Dong designed N1, N3, N5-tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (TT) and applied an orthogonal experimental design to investigate the impacts of formulation components on delivery efficiency. Lead materials comprised of TT, lipid/lipid-like nanoparticles (LLNs), and mRNA encoding human Factor IX (hFIX), fully recovered the level of human Factor IX to normal physiological values in FIX-knockout mice. In addition, he presented results demonstrating that these TT-LLNs were capable of effectively delivering Cas9 mRNA and guide RNA to the mouse liver for genome editing.

Jessica Madigan. Photo by Jerry Zon.

I’m pleased to say that a portion of Prof. Dong’s work was enabled by mRNA gifted by TriLink, and I am doubly pleased that TriLink presented two posters on its mRNA technology and products. One TriLink poster titled Key Critical Quality Attributes for the cGMP Production of Therapeutic Messenger RNA was presented by Jessica Madigan, pictured here, and Craig Dobbs presented a second TriLink poster titled Considerations for the Design and cGMP Manufacturing of mRNA, which featured a novel method for improved synthesis of 5’-cap analogs of mRNA that is referred to as CleanCap®.

Enhanced Delivery of mRNA-Protein Complexes

In the multi-talk Session on mRNA and CRISPR, Prof. Paula Hammond, Head of the Department of Chemical Engineering at Massachusetts Institute of Technology, gave a presentation titled Designer Polypeptides and Electrostatic Assembly for RNA-Protein Enhanced Delivery. The title alone piqued my interest because, in general, macromolecular self-assembly to form functional complexes in a predetermined manner is a trending topic in nucleic acid chemistry.

Prof. Hammond started off by saying that the traditional approach to mRNA delivery is the encapsulation of mRNA within a polycation. This approach requires mRNA to escape the polycation following endosomal uptake of the cargo, and to ultimately access the cytosol, where it can be translated. The translation step requires binding of the mRNA capping protein, EIF4E, Eukaryotic translation initiation factor 4E, and without this binding step, the mRNA is marked for degradation within the cell.

Photo by Jerry Zon

By contrast, Prof. Hammons has demonstrated that the pre-encapsulation of the EIF4E cap pre-complexed with based-modified (5’-methylcytidine and pseudouridine) mRNA—obtained from TriLink—can enhance mRNA translation within cells, multifold in comparison to release of mRNA alone. Furthermore, it was found that a unique series of polypeptides with variable charged side chain structures can enhance encapsulation of mRNA with EIF4E, with optimal systems yielding 70 to 80 times that of the mRNA alone. A key to these polymers is their ability to cooperatively bind RNA to the associated protein machinery needed for translation, in order to enhance translation.

More recently, Prof. Hammond has found similar kinds of enhancements for the delivery of siRNA via co-complexation with the Ago-2 protein to create a pre-assembled version of RISC complex. Finally, the layer-by-layer approach can be used to generate finely tuned release surfaces that can release small molecule, proteins, nucleic acids and other biologic drugs over sustained time periods, and with significant control of release characteristics. According to Prof. Hammond, this approach is particularly attractive for the delivery of proteins and nucleic acids such as siRNA.

Stereopure Phosphorothioate (PS)-Modified Antisense Oligonucleotides

In my recent blog I wrote about the highlights of the 23rd International Roundtable on Nucleosides, Nucleotides, and Nucleic Acids, and I commented on the virtually ubiquitous use of PS-modified linkages in all types of strategies for oligonucleotide-based therapeutics. I added that attention was now being given to stereoselective synthesis as a means of evaluating the influence of stereopure Rp or Sp linkages. Consequently, I and everyone else listened very attentively to the talk titled Stereochemical Control of Antisense Oligonucleotides Enhances Target Efficacy given by Chandra Vargeese, PhD, Senior Vice President Drug Discovery at Wave Life Sciences (Cambridge, MA).

Dr. Vargeese introduced the approach being used, and then presented data for antisense oligonucleotides shown here. These oligonucleotides were comprised of stereorandom or stereopure Rp (red ˄) or Sp ( black ˅) linkages at specific positions in a 20-base gapmer antisense nucleotide (gray filled ο) of unspecified sequence that was used to measure the kinetics of RNase H-mediated cleavage of complementary RNA as a model target.

Vargeese and coworkers Poster 30 titled Optimized, Stereopure Antisense Oligonucleotides Achieve Broad Tissue Distribution and Excellent Exposure, Enabling Potent and Durable Knockdown of Nuclear Malat1 in Mice and Nonhuman Primates. Photo by Jerry Zon.

Under the reported conditions, this construct gave initial kinetics that were 2-fold faster than the corresponding stereorandom antisense sequence. While this difference in efficiency was not very impressive, in my opinion, it was followed by in vitro results for free-uptake of stereopure and stereorandom antisense oligonucleotides against Malat1. In these experiments, the inhibitory concentration (IC50) values indicated 20-fold increased potency for the stereopure compound, which is quite impressive. Moreover, knockdown of Malat1 mRNA by the stereopure compound was about 10- to 100-times more effective than the stereorandom compound in mouse eye at 1 week.

Dr. Vargeese presented very intriguing data on uptake and distribution, which is not easily summarized here, but will hopefully be published with all of the above data in the near future alongside efficacy results for disease models in animals.

OTS Lifetime Achievement Award

Prof. Marv Caruthers award lecture. Taken from @OTSociety with its permission

The aforementioned synthesis of stereopure PS-oligonucleotides, which is enabled by phosphoramidite coupling followed by P3 → P5 conversion with a sulfurizing agent, is my segue into the OTS Lifetime Achievement Award. These year’s honoree was Prof. Marvin (“Marv”) Caruthers, whose talk was titled Chemical Synthesis of DNA/RNA and Biological Activity of Selected Analogues.

Marv began with his recollections of his lab’s development of the now universally employed phosphoramidite synthesis method, which he said was aimed at the younger members of the audience, jokingly adding that “oligos aren’t magically made by Federal Express.” Marv ended his talk with a description of how he has morphed his method to provide a wide variety of novel analogues. My blog about Marv’s marvelous contribution can be read here.

Delivery of Oligonucleotides to the Brain

At the risk of oversimplification, the human brain is sort of a “sanctuary” organ by virtue of a biochemical/vascular barrier (aka the blood brain barrier) that prevents or mutes continuous assaults, if you will, by chemicals or biochemicals that are harmful to the brain and its central nervous system (CNS). This makes delivering small drugs to the brain difficult, let alone relatively large single-stranded oligonucleotides. Consequently, current approaches for delivery of oligonucleotides to the brain have been largely restricted to using invasive intrathecal administration.

Blood brain barrier. Taken from Shutterstock. //  Intrathecal lumbar administration. Taken from Chung et al. (2016) PLOS One. Credit decade3D

Dr. Suzan Hammond. Photo by Jerry Zon.

By striking contrast to intrathecal administration of oligonucleotides, Dr. Suzan Hammond, pictured here, reported use of conventional systemic administration. Dr. Hammond is a postdoc with Prof. Matthew Wood in the Department of Physiology, Anatomy, and Genetics at the University of Oxford in the UK. She presented a poster titled Targeting the Brain and Spinal Cord with Antisense Oligonucleotides.

This work, which was carried out in collaboration with my long-time friend and oligonucleotide synthesis guru Prof. Michael Gait, employs a rather remarkable peptide-“oligonucleotide” conjugate. The “oligonucleotide” portion is a neutral-backbone, 6-membered ring morpholino (PMO) structure, which is quite unlike a deoxyribonucleotide, and has always been somewhat of an oddity in the oligonucleotide field. In any case, the PMO is conjugated to a PMO-internalizing peptide (Pip).

Motor neuron controls muscle movement. Taken from Shutterstock by Alila Medical Media

Interested readers can consult full details in a publication by Hammond et al. in the prestigious Journal of the Proceedings of the National Academy of Sciences. Dr. Hammond’s poster reports preclinical data that demonstrates potent efficacy in both the central nervous system (CNS) and peripheral tissues in severe spinal muscular atrophy (SMA), a leading genetic cause of infant mortality primarily due to lower motor neuron degeneration and progressive muscle weakness, which results from loss of the ubiquitous survival motor neuron 1 gene (SMN1).

Therapeutic splice-switching oligonucleotides (SSOs) modulate exon 7 splicing of the nearly identical SMN2 gene to generate a functional SMN protein. Peptide-PMOs yield SMN expression at high efficiency in peripheral and CNS tissues, resulting in profound phenotypic correction at doses an order-of-magnitude lower than required by standard naked SSOs.

As a follow-up to my question about future plans, Prof. Gait kindly provided the following reply by email:

“There is ongoing work in progress in Oxford, including a small spin off
company, to take the concept of peptide-PMO drugs towards clinical
trials in the next 2 or 3 years for one or more of the well-known
neuromuscular diseases, that include SMA. However, in the case of SMA, a
further grant-funded study is shortly to be underway to select a peptide
that might have a favourable toxicology profile, whilst maintaining
strong activity than the current peptides reported here, in the
expectation of reaching a suitable clinical candidate as soon as possible.”  

Concluding Impressions

Mid-morning and mid-afternoon coffee break networking was very enthusiastic. Photo by Jerry Zon

As a researcher who shared the initial vision of oligonucleotides as new therapeutics in the late 1970s—which faced far more naysayers than believers—I was more than gratified to be part of OTS 2018, and feel the positive “vibes” among nearly 600 participants, especially since this group included many young next-generation investigators, who represent the critical mass needed for future successes in this now firmly established field.

And speaking of the future, please note that in 2019 the 15th Annual OTS Meeting will be held in Munich, Germany.

Ich hoffe dich dort zu sehen!

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Given the difficulty of obtaining grant money for research, some of you will be pleased to know that the Ono Pharma Foundation sponsors the Breakthrough Science Initiative Awards Program. At OTS 2018, a representative of Ono Pharma gave the following information:

This program targets “groundbreaking work using synthetic oligonucleotides to gain valuable insights into molecular mechanisms, delivery strategies, or physiologic targets.” The grant is for $900,000 plus up to 15% indirect costs for 3 years of research, and there is no IP (intellectual property) obligation. Eligibility is for institutions in the US and Canada. Previous awardees are Jonathan Watts, PhD (Univ. of Mass.) in 2017, and William Mobley, MD, PhD (Univ. of Calif., San Diego).






CRISPR in the Clinic…Coming Soon

  • Trio of CRISPR Discoverers Awarded a $1 Million Kavli Prize
  • CRISPR Therapeutics, a Startup Company, Will Soon Start Clinical Trials
  • New Issue: Concerns for Cancer

Over the past few years, I have periodically blogged about CRISPR-based gene editing, which has been arguably the hottest trending topic in nucleic acid-targeted therapy for about the past five years or so. The catalyst for this burst of publications was a 2012 report in Science on a study led by Doudna and Charpentier (see below). The study focused on the potential utility of CRISPR-Cas9 for genome editing, and it currently has over 5000 citations in Google Scholar. There are ~7400 articles in PubMed indexed to CRISPR, and it is evident from my chart shown here that there is strong growth in the annual number of CRISPR publications in the PubMed database.

Number of CRISPR publications in PubMed

In May 2018, three pioneers in CRISPR technology—Emmanuelle Charpentier of the Max Planck Institute for Infection Biology in Berlin, Virginijus Šikšnys (see Footnote) of Vilnius University in Lithuania, and Jennifer Doudna of the University of California, Berkeley—were awarded the $1 million Kavli Prize in Nanomedicine. This highly prestigious prize from The Norwegian Academy of Science and Letters was awarded “for the invention of CRISPR-Cas9, a precise nanotool for editing DNA, causing a revolution in biology, agriculture, and medicine.”

Emmanuelle Charpentier, Virginijus Šikšnys and Jennifer Doudna (left to right). Taken from

As far as invention goes, there has been continued litigation, recently summarized in a GEN interview with law professor Jacob Sherkow titled CRISPR in the Courthouse. The University of California, Berkeley (UC) and the Broad Institute of MIT and Harvard are at odds over foundational patents covering CRISPR-Cas9. Interested readers should consult “late breaking news,” which covers the recent issuance of a US patent to UC and its partners.

Notwithstanding unresolved intellectual property matters, so-called “surrogate companies” for the holders of these key patents include Editas Medicine (MIT/Harvard), Caribou Biosciences/Intellia Therapeutics (UC and University of Vienna), and CRISPR Therapeutics (Emmanuelle Charpentier), the latter of which is the focus of the present blog. As you’ll read below, CRISPR Therapeutics is developing a “CTX001” approach for the treatment of Sickle cell disease and β-thalassemia for clinical trials this year, which is a highly anticipated milestone—both scientifically and commercially.

CRISPR Therapeutics CTX001

Sickle cell disease and β-thalassemia are caused by genetic mutations in the β-globin gene, which codes for the β subunit of hemoglobin that, as depicted below, is the oxygen carrying component of red blood cells. In these diseases, hemoglobin is missing or defective, which results in devastating medical problems. The approach developed by CRISPR Therapeutics is designed to mimic the presence of fetal hemoglobin (HbF; aka γ-globin) that is present in newborn babies. HbF is a form of hemoglobin that is quickly replaced by adult hemoglobin. However, in rare cases where HbF persists in adults, it provides a protective effect for those who have Sickle cell disease and β-thalassemia.

Taken from

CTX001 is an ex vivo therapy in which autologous (i.e. self-donated) cells are harvested directly from the patient. CRISPR Therapeutics then applies its gene-editing technology to the cells outside of the body, making a single genetic change designed to increase HbF levels in a patient’s own blood cells. The edited cells are then reinfused and are expected to produce red blood cells that contain HbF in the patient’s body, thus overcoming the hemoglobin deficiencies caused by these diseases.

The gene-editing mechanism for CTX001 presented by CRISPR Therapeutics at the American Society of Hematology (ASH) in December 2017 is depicted below. In researching this CRISPR-based mechanism, I found a publication by Bjurström et al. that helps to better understand this depiction. In brief, the zinc-finger transcriptional factor BCL11A has been shown to silence HbF genes in human cells during development, and thus directly regulates HbF switching.

Taken from CRISPR Therapeutics

BCL11A silences HbF by associating with other known γ-globin transcriptional repressors. The gene binds to the locus control region as well as other intergenic sites, which prevents the interaction between the locus control region and the HbF globin gene required for fetal globin expression. Using a guide RNA and Cas9 to enable permanent site-specific genome engineering through a DNA repair pathway, knockdown of the BCL11A gene can be an effective strategy for reactivating HbF and restoring functional erythrocytes.

The aforementioned ASH presentation by CRISPR Therapeutics also includes an overview of Sickle cell disease and β-thalassemia, as shown here. According to an informative historical article that I found, Sickle cell disease and β-thalassemia are related genetic disorders that can cause fatigue, jaundice, and episodes of pain ranging from mild to very severe. They are inherited, and usually both parents must pass on an abnormal gene in order for a child to have the disease. Much more genetic information on these two disorders is available on the NIH Genetics Home Reference.

Taken from CRISPR Therapeutics

CRISPR Therapeutics Clinical Studies Status

The December 2017 ASH presentation by CRISPR Therapeutics received widespread media coverage that heralded the highly anticipated “bench-to-bedside” transition for CRISPR technology. CTX001 was able to efficiently edit the target gene in more than 90 percent of hematopoietic stem cells to achieve about 40 percent of HbF production, which investigators believe is sufficient to improve a patient’s symptoms. Study results also showed that CTX001 affects only cells at the target site and that it has no off-target effects on hematopoietic stem cells, thereby appearing to be a safe potential treatment.

These positive results prompted CRISPR Therapeutics to start a collaboration with Vertex Pharmaceuticals to develop and commercialize CTX001 treatment of Sickle cell disease and β-thalassemia. It was also announced that CRISPR Therapeutics and Vertex are planning to submit an investigational new drug (IND) application to the Food and Drug Administration (FDA) to start a Phase 1/2 clinical trial in Sickle cell disease in the United States in 2018. In addition, CRISPR Therapeutics also submitted a clinical trial application (CTA) for CTX001 to advance into a Phase 1/2 clinical trial in patients with β-thalassemia in Europe in 2018. This trial will evaluate the safety and effectiveness of CTX001 in adult patients with transfusion-dependent β-thalassemia.

After the above announcement, News Atlas reported that the FDA placed a clinical hold on this Phase1/2 trial of CTX001 pending, according to CRISPR Therapeutics, ‘the resolution of certain questions that will be provided by the FDA as part of its review of the IND.’

Concerns for Cancer

In studies published in June 2018 in venerable Nature Medicine, researchers from Sweden’s Karolinska Institute and, separately, Novartis, found that cells whose genomes are successfully edited by CRISPR-Cas9 have the potential to seed tumors inside a patient. CRISPR-Cas9 works by cutting both strands of the DNA double helix. That “injury” causes a cell to activate a gene called p53, which has been called the “Guardian Angel of the Genome” and is the most studied of all human genes, which you can read about in one of my previous blogs.

Whichever action p53 takes, the consequence is the same: CRISPR doesn’t work as intended because the genome edit is mended, or the cell dies. The flip-side of p53 repairing CRISPR edits, or killing cells that accept the edits, is that cells that survive with the edits do so because they have a dysfunctional p53. The reason why that could be a problem is that p53 dysfunction can cause cancer. The p53 gene is reported to be the most frequently mutated gene in human cancer: about 50% of all human cancers have lost p53 or express an inactive, mutant p53.

As a result, the Novartis paper concludes that “it will be critical to ensure that [genome-edited cells] have a functional p53 before and after [genome] engineering.” The Karolinska team warns that p53 and related genes “should be monitored when developing cell-based therapies utilizing CRISPR-Cas9.”

An article in quotes the CEO of CRISPR Therapeutics, Sam Kulkarni, as saying that these p53 findings are “something we need to pay attention to, especially as CRISPR expands to more diseases. We need to do the work and make sure edited cells returned to patients don’t become cancerous.”

Closing Comments

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Many years ago, I was among the early investigators of antisense therapeutics, which at the time was viewed as a new paradigm that would enable faster bench-to bedside, compared to traditional small molecule drug development. In reality, the antisense approach encountered unforeseen complications and required ~30 years of development to reach demonstrable clinical utility, which I previously wrote about in another blog. Short-interfering RNA (siRNA)-based therapeutics also encountered similar struggles.

While past history is not a predictor of the future, in my humble opinion, CRISPR-based clinical strategies will continue to have to deal with unexpected issues, such as the above p53 situation. While I remain hopefully optimistic about future clinical successes for CRISPR, I won’t be surprised if some of these achievements come slower than currently anticipated.

As usual, your comments are welcomed.


According to June 8, 2018 Science News at a Glance, Virginijus Šikšnys, whose role in the invention of the revolutionary genome editor CRISPR has often been overlooked, received some vindication when he was named a co-winner of the prestigious Kavli Prize in Nanoscience. Šikšnys will share the $1 million award with Doudna and Charpentier, who have received far more attention. Šikšnys first showed that the CRISPR system could be transferred from one bacterium to another. And like Doudna and Charpentier, he independently designed a way to steer the CRISPR complex to specific targets on a genome, which he called “directed DNA surgery.”




Chinese Scientists to Pioneer First Human CRISPR Clinical Trial

  • Chinese Team Begins First CRISPR-Based Anticancer Immunotherapy Clinical Study
  • US Consortium’s CRISPR Clinical Study OK’d by NIH Panel
  • Survey Indicates More Worry than Enthusiasm for Gene-Editing in Babies
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Regular readers of my blog will recall a number of previous postings on gene editing using CRISPR-Cas9 (aka CRISPR), which has rapidly become the hottest trend in nucleic acid-based biotechnology and medical research. That opinion is partly based on the fact that this relatively new technology has already amassed thousands of publications, including 1,250 in 2015 alone! Additional evidence follows from the numerous start-up and established biopharma companies pursuing CRISPR-based therapeutics, which is “the biggest biotech discovery of the century” according to one report.

Now, even more exciting developments for CRISPR are on the horizon. Chinese researchers are poised for the first human clinical trial using CRISPR, and an analogous study in the US awaiting FDA approval. Both of these trials involve forms of cancer immunotherapy, which is a very hot field in itself, and is briefly introduced in the next section.

Cancer Immunotherapy

Cancer immunotherapy is the use of the immune system to treat cancer either actively, passively, or by a combination of these approaches, which exploit the fact that cancer cells often have macromolecules on their surface that can be detected by the immune system (aka tumor-associated antigens, TAAs). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs, whereas passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines.

Numerous antibody therapies have been approved by the FDA and other regulatory authorities worldwide. In contrast, active immunotherapies, which usually involve the removal of immune cells from the blood or from a tumor for reintroduction to the patient, have lagged in such approvals. In fact, the only US-approved cell-based therapy is Dendreon’s Provenge®, for the treatment of prostate cancer. A recent series of NY Times articles highlights some powerful examples of cancer immunotherapy, especially from the perspective of interviews with patients and their physicians.

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Taken from

For the purpose of this blog, I’ll add that adoptive T-cell therapy is a form of passive immunization by the transfusion of T-cells, which are found in blood and tissue and usually activate when they find “foreign” pathogens. These pathogens can be either infected cells, or antigen presenting cells present in tumor tissue, where they are known as tumor infiltrating lymphocytes. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumor death.

As depicted below, T-cells specific to a tumor antigen can be removed from a tumor sample or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the resultant cells being reinfused. Importantly, activation can take place through genetic engineering, such as gene editing by CRISPR.

Cancer specific T-cells can be obtained by fragmentation and isolation of tumor infiltrating lymphocytes, or by genetically engineering cells from peripheral blood. The cells are activated and grown prior to transfusion into the recipient (tumor bearer). Taken from

Cancer specific T-cells can be obtained by fragmentation and isolation of tumor infiltrating lymphocytes, or by genetically engineering cells from peripheral blood. The cells are activated and grown prior to transfusion into the recipient (tumor bearer). Taken from

CRISPR Goes Clinical in China

As I write this post, Chinese scientists at Sichuan University’s West China Hospital in Chengdu will reportedly become the first in the world to inject people with cells modified using the CRISPR gene-editing method. This landmark trial will involve patients with metastatic non-small cell lung cancer who have not responded to chemotherapy, radiation therapy and/or other treatments.

The team will extract T cells from the blood of the trial participants and then use CRISPR to knock out a gene encoding a protein called PD-1 that normally regulates the T cell’s immune response to prevent it from attacking healthy cells. As depicted below, this so-called checkpoint has been targeted by small molecule inhibitors (anti PD-1), but with somewhat limited success so far—notably including failure of a would-be billion-dollar drug (Opdivo®) recently reported by Bristol-Myers Squibb. Nevertheless, the presently envisaged PD-1 gene-edited cells will be amplified in the lab and re-introduced into the patient’s bloodstream to circulate and, hopefully, attack the cancer.

Taken from

Taken from

Potential Concerns of CRISPR Clinical Trials

While the Chinese trial may be groundbreaking, it is not without risk. There is concern that the edited T-cells could attack normal tissue as it’s well documented that CRISPR can result in gene edits at the wrong place in the genome. To help mediate this risk, the T-cells will be validated by biotechnology company MedGenCell—a collaborator on the trial—to ensure that the correct gene is knocked out before the cells are re-introduced into patients.  Since the primary purpose of this phase one trial is to determine if the approach is safe, participants will be closely monitored for side effects, and researchers will pay close attention to biological markers in the blood.

Some may say that this type of trial in humans is premature, but China has always tried to be a leader in CRISPR research. Carl June, a clinical researcher in immunotherapy at the University of Pennsylvania in Philadelphia explains, ‘China places a high priority on biomedical research.’ And Tetsuya Ishii, a bioethicist in Japan, is quoted in Nature magazine as saying that ‘When it comes to gene editing, China goes first.”

Initial CRISPR Clinical Trial in USA Awaiting Review Board and FDA Approvals

While the Chinese team seems positioned to be the first to conduct a CRISPR clinical trial, the US isn’t far behind. As reported earlier this summer, the Recombinant DNA Research Advisory Committee (RAC) at the NIH has approved an analogous—but genetically more complex—proposal to use CRISPR-edited T cells for cancer immunotherapy. The RAC reviews all proposals for human trials involving modified DNA that are conducted in the United States, and the investigators now have to convince review boards at their own institutions, and the FDA, to allow the trial. This will hopefully be done by the end of 2016.

The proposed trial is a collaborative effort involving the University of Pennsylvania, M.D. Anderson Cancer Center in Texas, and the University of California, San Francisco. This trial will be funded by a $250-million immunotherapy foundation formed in April 2016 by former Facebook president Sean Parker. According to a news item in Nature, The University of Pennsylvania will produce the edited cells, and will recruit and treat patients alongside the collaborating medical centers in Texas and California.

In this study, researchers will perform three CRISPR edits with T cells from patients with several types of cancers: 1. a gene for a protein engineered to detect and target cancer cells will be inserted, 2. a natural T-cell protein that could interfere with this process will be removed and 3. the gene for a protein that identifies the T cells as immune cells and prevents the cancer cells from disabling them will be removed. The researchers will then infuse the edited cells back into the patient.

Both of the studies being performed by the Chinese and US teams are very exciting to me. I will be paying close attention to the progress of these studies over the next few months and hope to report back to you with some very interesting and ground breaking results.

Survey Indicates Public Concern in the US About Gene Editing in Babies

A Pew Research Center pole focusing on concerns about gene editing was recently reported in Nature. The poll surveyed more than 4,700 people in the United States and the results indicate that more people are concerned than enthusiastic about gene editing.

The quadrants below (from 0-100%) show responses to the question, “How worried or enthusiastic do you feel about gene editing to reduce disease risk in babies?”

Taken from

Taken from

While these findings speak for themselves, I hasten to add that this same pole also asked “Have you heard or read about this topic?” The following replies (in quadrants from 0-100%) indicate that a substantial percentage of respondents who have not at all read about gene editing nevertheless expressed either worry or enthusiasm for gene editing. Ditto for those who read a little about gene editing.


It would seem, then, that we should take this poll with a grain (or two) of salt as it apparently includes a substantial amount of uninformed opinion. This is not too surprising given the complex nature of gene editing and it’s relatively new position in research. We can see from the results of this poll that a significant amount of education may be needed to garner increased public support for gene editing. Hopefully, the results of the studies highlighted above will begin to pave this road.

Hope for the Best but Expect Complications

This section heading, which summarizes my parting comments about the advent of CRISPR-enabled clinical trials, is based upon my having participated in several decades of research on nucleic acid-based concepts for radically different approaches to traditional small-molecule therapeutics. First was the idea of using chemically modified antisense oligonucleotides to block gene expression, which encountered non-specificity issues, low potency, delivery, and a host of other issues that took two decades to sort through. Then there was the idea of using chemically modified short-interfering RNA (siRNA) for modulating gene expression via RNA interference (RNAi). This, too, encountered similar problems in reaching the clinic. Ditto for anti-microRNAs (antagomirs).

I’m hoping that CRISPR-based therapies meet with success far faster—and prove to be affordable to society. I won’t, however, be surprised if progress is slow—and quite expensive.

Are you excited about potential CRISPR-based therapies? Do you have concerns about their safety and efficacy? Do you believe the general public is ready to accept gene editing therapies? Please share your thoughts as your comments are always welcome.

DIY CRISPR Kit – Door to Democratization or Disaster?

  • Gene Editing with CRISPR is All the “Buzz”
  • Low-Cost CRISPR Kit Being Sold to DIY “Biohackers”
  • What is the Balance Between Democratization and Preventing Disaster?

The dictionary definition of democratization is the transition to a more democratic political regime. Since democracy emphasizes the role of individuals in society, democratization is generally perceived to be good. This political concept of democratization is being increasingly morphed, if you will, to describe the transition of science and technology from trained specialists in traditional labs to any individual, anywhere—including someone’s kitchen table.

Taken from

Taken from

Lest you get the impression I’m an elitist, and not in favor of fostering better understanding—and appreciation—of science by non-scientists everywhere, I definitely am not. I want the value of science to be widely appreciated. Even if I weren’t of that opinion, democratization of science and technology is already evident in this exemplary cartoon indicating how DNA is now familiar to virtually everyone. But I digress…

Taken from


It is evident that molecular biology has also undergone democratization based on emergence of so-called “do it yourself” (DIY) advocates of biology (DIY-BIO), which on the surface seems like a good thing. But, as I’ll expand upon below, DIY-BIO has morphed in a way which has elevated concerns that a well-intentioned DIY aficionado anywhere can now access genetically powerful CRISPR reagents that might inadvertently unleash a harmful home-made organism.


First off, I should note that gene editing by CRISPR—thankfully short for “clustered regularly-interspaced short palindromic repeats”—actually involves another component named Cas9—short for CRISPR associated protein 9. Cas9 is an enzyme that recognizes single guide RNA (sgRNA) hybridized to one strand of specifically targeted DNA via the 5’end of sgRNA, as depicted in green in the mechanism below. The remaining sgRNA has a double-stranded “stem” (black, red) and loop (purple) internal structure, and a 3’ end with several stem-loop structures (red).

Taken from

Taken from

The scissors indicate Cas9 cutting both strands of DNA, which thus allows for insertion of so-called donor DNA and, consequently, enabling a variety of genetic manipulations in plants, bacteria, human or animal cells. Chemically synthesized sgRNA that target any gene of interest can be readily designed for purchase, along with Cas9 in the form of biosynthetic Cas9 mRNA encoding this necessary protein component.

CRISPR’s importance as an emerging, useful tool for gene editing is evident from the number of publications in PubMed that have approximately doubled each year since the seminal to give an estimated 2,500 publications indexed to CRISPR as a search term. Unfortunately (but perhaps not surprisingly given the billion-dollar implications), there is an ongoing dispute over inventorship involving the Broad Institute (see Feng Zhang patent), the University of California, and the University of Vienna.

Biohacker Promotes DIY CRISPR Kit

Josiah Zayner (Taken from

Josiah Zayner (Taken from

As mentioned in the introduction, self-proclaimed “biohackers” who are avid fans and practitioners of DIY molecular biology, have been busily “doing their thing” for some time now without much cautionary publicity. That’s changing, however, as a result of the advent of CRISPR together with relatively easy access to its sgRNA and Cas9 reagents. One case in point involves Josiah Zayner, who has a PhD from the Department of Biochemistry and Molecular Biophysics at the University of Chicago and now lives in the San Francisco Bay Area.

Zayner’s online biographical sketch states that he is “very active in Biohacking and DIY Science and run[s] an online Biohacking supply store The ODIN.” By visiting the website for The ODIN, which reportedly raised $65,000 by crowdfunding online via Indiegogo, you’ll find various items for conducting molecular biology experiments, along with an “about” page stating that “smaller groups of people, small labs or even DIY Scientists on their own can do amazing things if they have access to resources that are normally only available to large heavily funded labs and companies.”

While this seems all fine and good is some ways, the item offered by The ODIN that has led to controversy is the first-ever DIY kit for CRISPR. This, according to an article in The Mercury News, “raises the specter—deeply troubling to some experts—of a day when dangerous gene editing is conducted far from the eyes of government regulators, posing risk to the environment or human health”.

The article goes on to quote one expert who said The ODIN kit is sold for manipulating yeast and could never be used to alter human genes, while another expert cautioned that the kit can teach basic principles to do so with appropriate modifications. Another problem is inadvertent conversion of yeast into a harmful microorganism that might be accidentally spread.

Taken from

Taken from

While I share these concerns, it will be virtually impossible to prevent individuals or small groups intent on nefarious activities using CRISPR technology. On the other hand, I have to admit that I would be very concerned if I were living next door or otherwise nearby Josiah if he is indeed practicing what he’s preaching, so to speak, using CRISPR in his kitchen as pictured right.

CRISPRized Plants, Too

If you think that DIY is a passing fad with few devotees, think again. Aside from the main DIY-BIO website that you can peruse, a recent online article in Fusion talks about a couple of DIY enthusiasts doing things that make the hairs on my neck stand up, as the saying goes. For instance, David Ishee, a 30-year-old Mississippi resident who never attended college, does at-home experiments in his shed using online kits for growing plants, but will now use CRISPR to carry out gene editing.

Ishee reportedly will use software like DeskGen that advertises its “on-demand CRISPR libraries” for gene editing, and is quoted as saying “That gives me a lot of new options. Up until now, all the genetic edits I’ve made have been limited to plasmids and unguided genomic insertions. That limits the kinds of cells I can work with and the types of work I can do.”

So what will Ishee do? The answer is that nobody but he knows. If his genetically edited plants grow and seeds get carried by the wind, they could someday end up in your backyard. What then? Who knows? Could be creepy.

Possibly harmful, irreversible consequences of completely democratized CRISPR are completely unknown. Therein lies the essence of the problem that has many experts quite concerned, as reported in Fusion. I share that concern.

Parting Shot

In closing this brief story about DIY synthetic biology using CRISPR, I must say that I wish journalists writing for newspapers and other media would stick to news that is factual and not interpreted for commentary that is flat out wrong or intentionally provocative. My case in point is the following big font, bold letters headline:

“Finally, your chance to play God!”

This was used by to recycle the aforementioned piece by The Mercury News. Shame on for this misleading and totally wrong exclamation. But I digress…

I would greatly appreciate knowing your thoughts about DIY CRISPR by sharing them here as comments.

Zon on Zon’s Zebrafish

  • Leonard Zon Uses Zebrafish to “Fish” for Candidate Drug Compounds
  • Two Candidate Drugs are in Clinical Trials for Cancer Treatments
  • Zon Interviews L. Zon
Leonard I. Zon, M.D. Taken from

Leonard I. Zon, M.D. Taken from

The first time I was asked if I was related to the scientist Leonard Zon, I honestly had to reply that I didn’t know, and out of curiosity later looked up his publications, which were quite numerous for a then newish investigator. His current biosketch expertise includes pioneering research in the new fields of stem cell biology and cancer genetics. Dr. Leonard I. Zon is the Grousbeck Professor of Pediatric Medicine at Harvard Medical School, an Investigator with the Howard Hughes Medical Institute, and Director of the Stem Cell Program at Children’s Hospital Boston—that’s impressive!

I found Leonard Zon’s unusual zebrafish-based research and accomplishments therefrom definitely blogworthy, and the coincidences of both our surnames and involvement in science are kind of an unusual “double-doppelgänger,” if you will. In any case, it’s always a surprise to meet your double, even if only in name and profession.

A few regular readers of my blog will likely smile and think that Zon on Zon’s Zebrafish is yet another instance of my penchant for alliteration. However, reading the following snippets about Leonard Zon’s clever—dare I say zany—use of zebrafish for his research will illustrate why they are unusual, interesting and commercially viable.

On the other hand, I must admit that I too smiled at the thought of this unique opportunity to post an interview of one Zon by another Zon, which reminded me a bit of Zappa Plays Zappa. But I digress…

Zebrafish as a Model for Organogenesis

Leonard Zon currently has nearly 250 research publications listed in PubMed, which is a large number by any measure, but that’s even more impressive when you take into account that his first was in 2002—only 14 years ago. That translates to an average of about 3 publications every 2 months each year!

Zon’s inaugural publication in 2002 was an in-depth review in venerable Science in which the abstract presciently reads in part as follows:

Organs are specialized tissues used for enhanced physiology and environmental adaptation. The cells of the embryo are genetically programmed to establish organ form and function through conserved developmental modules. The zebrafish is a powerful model system that is poised to contribute to our basic understanding of vertebrate organogenesis. This review develops the theme of modules and illustrates how zebrafish have been particularly useful for understanding heart and blood formation.

As will be elaborated below, Zon’s most recent publication in 2016—also in Science—has extended the zebrafish model to now include melanoma. If you’re asking yourself, why use zebrafish, the answer is partly due to convenience derived from the unique features and accelerated life cycle of zebrafish.

Zebrafish life cycle. Taken from

Zebrafish life cycle. Taken from

Seen right, these advantages include its small size, easy care, and rapid generation time. In addition—and very importantly—the embryos and growing zebrafish are transparent, allowing for continuous observation of developing organs under the light microscope.

Mutagenesis screens allow examination of defects in early organogenesis and late organ function. These many advantages of investigating zebrafish—and Zon’s huge facility comprising thousands of tanks—are nicely explained and shown in a video, which I found well worth viewing. The video also includes Zon’s specially bred, virtually transparent species of zebrafish named—humorously—Casper, after Casper the Friendly Ghost. Pictures below are (a) the transparent Casper zebrafish; (b) the non-transparent wild-type zebrafish; and transparent Casper the Friendly Ghost, which brings back my childhood memories. But again I digress…let’s get back to science!

(a) Casper, (b) wild type zebrafish. Taken from Transparent Casper the Friendly Ghost. Taken from

(a) Casper, (b) wild type zebrafish. Taken from Transparent Casper the Friendly Ghost. Taken from

Aided by the availability of DNA sequence information for the zebrafish genome, researchers have published ~2,000 (!) reports dealing with antisense gene “knockdown” using phosphorodiamidate oligonucleotides. By numerical coincidence, the first such report appeared in 2000, and was prophetically entitled Effective targeted gene ‘knockdown’ in zebrafish.

Taken from

Taken from

As shown below, these rather unusual oligonucleotides—dubbed “morpholinos” by resemblance of the 6-membered ring to morpholine—can be injected directly into zebrafish embryos. Interested readers can consult a detailed “how to” guide on use of morpholinos in zebrafish.

Going forward, however, I expect that uber-hot CRSPR/Cas9 gene editing will be widely adopted, based on the titles of these two pioneering publications in 2016:

Stem Cells and Beyond

Now that we know a bit about Zon’s zebrafish and how to knockdown or edit a gene for functional genomics (aka “gene functionation”), let’s zero in on what Zon studies and how he does that. In a nutshell, the overarching science in Zon’s lab deals with stem cells, which are undifferentiated cells that can differentiate into specialized cells, as well as divide to produce more stem cells, as depicted below.

Taken from

Taken from

According to Zon’s website, the hematopoietic system that forms various types of blood cells is an excellent model for understanding tissue stem cells. This conceptual relationship is very important because it provides insight to cell differentiation regulation, and involvement in aging, disease, and oncogenesis. In addition, better understandings of the regulation of hematopoietic stem cell biology and lineage differentiation improves diagnosis and treatment of human hematopoietic disorders (aka “blood cancers”) and bone marrow transplantation therapies.

Differentiation of different blood cells from hematopoietic stem cell to mature cells. Taken from

Differentiation of different blood cells from hematopoietic stem cell to mature cells. Taken from

From Zebrafish to Clinic

Scientific theory is nice, but success is best, and Leonard Zon’s theory of using zebrafish to manipulate human stem cells for discovering therapies seems indeed to be headed for success, according to an article in the Harvard Gazette.

Zon and others at the Harvard Stem Cell Institute (HSCI) have published initial results of a Phase Ib safety study wherein 12 adult patients undergoing umbilical cord blood transplantation received two umbilical cord blood units, one untreated and the other treated with the small molecule 16,16-dimethyl prostaglandin E2 (dmPGE2). This molecule had been found in Zon’s zebrafish screen, which I’ll outline below.

Fate Therapeutics, a San Diego-based biopharmaceutical company of which Zon is a co-founder, sponsored the investigational new drug (IND) application, under which the aforementioned clinical program was conducted, thus translating his research findings from the laboratory—dare I say tank—into the clinic.

Zon’s Zebrafish Yield New Approaches to Treat Muscular Dystrophies

According to NIH’s National Institute of Neurological Disorders and Stroke website, muscular dystrophies (MD) are a group of more than 30 genetic diseases characterized by progressive weakness and degeneration of the skeletal muscles that control movement. It adds that there is no specific treatment to stop or reverse any form of MD. Consequently, MD is a compelling target for new drug discovery, and Zon’s zebrafish are being used for such discovery in the following way.

Each zebrafish produces about 200 (!) eggs per week, so Zon’s lab collects and deposits a single egg into each well of a multi-well plate for individual but parallelized treatment with individual chemicals to screen for effects on differentiation, thus screening many compounds in a speedy manner.

Zon and coworkers concluded that “these studies reveal functionally conserved pathways regulating myogenesis across species [zebrafish, mouse, and human] and identify chemical compounds that…differentiate human iPSCs into engraftable muscle.”

Let’s hope that human clinical trials using this novel therapeutic approach enabled by Zon’s zebrafish soon prove successful for treatment of MD.

Zon’s Zebrafish Also Enable Elucidation of Melanoma Development

According to a fact page by the American Cancer Society, skin cancer is the most common of all cancers. About 3.5 million cases of basal and squamous cell skin cancer are diagnosed in this country each year. Melanoma, a more dangerous type of skin cancer, will account for more than 73,000 cases of skin cancer in 2015.

Zon and a large group of 18 coworkers have recently reported in venerable Science work that has been heralded in a New York Times article. This study is very comprehensive and involves lots of “heavy duty” molecular and cellular biology, which you can read in detail in the aforementioned linked article. Snippets of the key findings are as follows.

  • Benign melanocytic skin cells carry oncogenic BRAF-V600E mutations and can be considered a “cancerized” field of melanocytes, but they rarely convert to melanoma.
  • In an effort to define events that initiate cancer, they used a melanoma model in the zebrafish in which the human BRAF-V600E oncogene is driven by the melanocyte-specific mitfa
  • When bred into a p53 mutant background, these fish develop melanoma tumors over the course of many months.
  • The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and is specifically reexpressed only in melanoma tumors, making it an ideal candidate for tracking melanoma from initiation onward.
  • As show below, they developed a crestin:EGFP reporter that recapitulates the embryonic neural crest expression pattern of crestin and its expression in melanoma tumors.
  • They show through live imaging of transgenic zebrafish crestin reporters that within a cancerized field (BRAFV600E-mutant; p53-deficient), a single melanocyte reactivates the NCP state, and this establishes that a fate change occurs at melanoma initiation in this model.
Taken from Zon and coworkers Science 2016.

Taken from Zon and coworkers Science 2016.

Zon Interviews L. Zon

Dr. Zon and some of his 4,000 zebrafish tanks. Taken from

Dr. Zon and some of his 4,000 zebrafish tanks. Taken from

After researching Leonard Zon’s aforementioned unique—and promising—use of zebrafish to advance basic science and discover new therapies, I contacted him by email to “interview” him, regarding several points. My questions (JZ) and his answers (LZ) are as follows.

JZ: Aside from the cord blood clinical trial mentioned in the Harvard Gazette, are any other human clinical trials being carried out based on your zebrafish findings?

LZ: We have had two chemicals discovered in zebrafish, and ultimately went to a clinical trial. The first was a di-methyl form of PGE2 for cord blood transplantation for leukemia. The second was leflunomide, an arthritis drug that paused transcription in neural crest cells and is being evaluated for metastatic melanoma.

JZ: Is Fate Therapeutics your only startup company?

LZ: I started Scholar Rock about 3 years ago. This company is targeting the TGF-B family of ligands. Has about 25 employees, and is in Cambridge. I am about to start a third company.

JZ: Is your zebrafish method for screening chemicals patented?

LZ: We have patents on several screening methods, but in general we patent the chemicals we find.

JZ: Zebrafish offer many reported advantages for your kind of research, but what is a primary disadvantage?

LZ: The major disadvantage of the zebrafish is that the system occasionally lacks definition. For instance, in the blood system, we have one monoclonal antibody against one epitope. We really need to create reagents for the field that brings it in line with other systems such as mice and humans.

JZ: How many tanks and zebrafish are maintained in your two labs?

LZ: We have 4000 tanks and about 300,000 fish.

JZ: Did you name your transparent Casper zebrafish after Casper the Friendly Ghost?

LZ: Absolutely.

In closing, I should add that the huge amount of information on zebrafish as a model organism for human disease and drug discovery from many labs has been centralized and organized in a database that is available through The Zebrafish Information Network (ZFIN) for researchers to share at the ZFIN Community Wiki.

I hope that you found this blog interesting, and I welcome your comments.

Joe Zon with some of his famous guitars at NAMM Show 2015. Taken from

Joe Zon with some of his famous guitars at NAMM Show 2015. Taken from


Truth be told, compared to being asked if I’m related to Leonard Zon, I’m more frequently asked if I’m related to Zon guitars, which apparently are quite well known, and are produced in Redwood City, CA by Joe Zon, who is pictured below. My reply to that frequent question is that I don’t know if I’m related, but will someday look into that, as well as whether I’m distantly related to Leonard Zon.