CRISPR-C2c2 Update: Powerful New Diagnostic Method Using CRISPR

  • CRISPR Craze Continues Led in Part by Wunderkind Feng Zhang
  • Nonspecific CRISPR-C2c2 “Collateral” Cutting Channeled into Diagnostics
  • Turning Biochemical “Lemons to Lemonade”

This blog post is about a new and powerful diagnostic approach based on CRISPR, which I’ll get to below. But first I’d like to point out several reasons why this is an especially interesting development:

  • Taken from spyhollywood.com

    Any new method using CRISPR is more “sizzle” for this “super-hot” technology

  • Feng Zhang, the 32-year-old author of this publication, is regarded as a wunderkind
  • My past blog post on CRISPR-C2c2 “collateral” cutting noted possibilities for turning “lemons into lemonade”
  • Such “lemonade” has emerged as a diagnostic cleverly acronymized as SHERLOCK

So, without further ado, here’s a brief recap of CRISPR-C2c2 as an intro to SHERLOCK (think Holmes), followed by why this acronym is apropos for a diagnostic method that magnifies detection, in this case Zika virus, about which I’ve previously commented. 

What’s CRISPR-C2c2?

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (CRISPR-Cas) adaptive immune systems in microbes contain endonucleases that can be leveraged for CRISPR-based gene editing using targeted CRISPR RNAs (crRNAs), as I’ve outlined in a previous blog post. While such Cas enzymes target DNA, others target RNA and function as RNA-guided RNases, which was reported by Feng Zhang and collaborators in Science last year in an article titled C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector.

My blog post about that C2c2 publication provides more information, but the main point related to the present blog post about diagnostics is that after recognition of its RNA target, activated C2c2 engages in nonspecific (aka “collateral”) cleavage of nearby non-targeted RNAs. This post-cleavage non-specificity can be transformed, if you will, from something “bad” to something “good” in terms of mechanisms, which I described as being akin to converting ‘lemons into lemonade’. You’ll now see how SHERLOCK is such ‘lemonade’ concocted by Zhang and coworkers.

What is SHERLOCK?

Taken from US 20140199245 A1

Before answering this question, and not to confuse you, I need to first mention that the nuclease originally called C2c2 was renamed Cas13a to be more systematic, and will be referred to herein as such. Having said that, Cas13a can be reprogrammed with crRNAs to provide a platform for specific RNA sensing. Molecular recognition of an intended RNA target by crRNA in vitro serves as a sequence-specific “trigger” to activate Cas13a’s nonspecific, “collateral” cleavage of labeled RNA reporters. Cleavage of these fluorescently labeled, internally quenched RNAs leads to unquenching and fluorescent light emission for detection, as depicted below for a generic RNase.

Now that you have a sense of how CRISPR-Cas13a can generate a signal, you’re probably asking ‘What is SHERLOCK?’ It’s an acronym coined by a large team lead by CRISPR genome-editing pioneer Feng Zhang and bioengineer James Collins, both from the Broad Institute. SHERLOCK stands for High-sensitivity Enzymatic Reporter UnLOCKing, which is a recently published new method for sequence-specific detection of DNA or RNA in any sample of interest. This general utility represents a major advance in CRISPR-based diagnostics.

Feng Zhang. Taken from be.mit.edu // James Collins. Taken from achetron.com

As depicted below, sample prep workflows allow for input of either double-stranded DNA (dsDNA) or RNA, which are first amplified by recombinase polymerase amplification (RPA) directly or as cDNA, after reverse transcription (RT-RPA), respectively. In either case, further amplification utilizes T7 transcription into RNA (blue = target RNA; yellow = non-target RNA). Following cleavage of target RNA by Cas13a-crRNA, a commercially available “cleavage reporter” (i.e. internally quenched) RNA undergoes “collateral” cleavage to generate a reporter signal (i.e. fluorescence; pictured below) for real-time detection of the target.

Taken from Feng Zhang, Collins & coworkers Science (2017)

An advantage of RPA is that it is performed isothermally, which allows simplification of the detection device by eliminating the need for a thermal cycler, thus lending to incorporation into small, portable point-of-care (POC) systems (about which I previously commented several times). The abovementioned RPA/T7 sample prep methodology was shown to have attomolar (aM, 10−18 mol/L) sensitivity in model systems, and was next investigated for sensitivity and specificity of virus detection, as well as simplification of reagent format.

Zhang, Collins & coworkers constructed lentiviruses harboring genome fragments of either Zika virus (ZIKV) or the related flavivirus Dengue (DENV), and showed that SHERLOCK detected viral particles down to 2 aM could discriminate between ZIKV and DENV. To explore the potential for POC use of SHERLOCK with paper-spotting and lyophilization (aka freeze-drying), they first demonstrated that Cas13a-crRNA complexes lyophilized and subsequently rehydrated could detect 20 femtomolar (fM, 10−15 mol/L) non-amplified RNA, and that target detection was also possible on glass fiber paper.

The other components of SHERLOCK, namely the RPA reagents and T7 polymerase, were already known to be amenable to freeze-drying and storage at ambient temperatures. In combination, freeze-drying and paper-spotting the Cas13a detection reaction resulted in comparable levels of sensitive detection of RNA as aqueous reactions. Although paper-spotting and lyophilization slightly reduced the absolute signal of the readout, SHERLOCK could readily detect mock ZIKV virus at concentrations as low as 20 aM. Most importantly, SHERLOCK detected ZIKV in clinical isolates (ZIKV RNA extracted from patient serum or urine samples and reverse transcribed into cDNA) could be detected at concentrations down to 1.25 × 103 copies/mL (2.1 aM), as verified by qPCR.

Concluding Comments

If you reflect upon the schematic for SHERLOCK, you’ll note that the input can be either DNA or RNA, which get amplified to produce many copies of RNA that serve as substrates for cleavage by Cas13a-crRNA, thus inducing collateral cleavage of reporter RNA to produce a detectable signal. Lest you think SHERLOCK is too costly to be practical, its developers provide a detailed cost accounting that estimates $0.61 per test, which you’re welcome to compare with your cost of conventional qPCR. I’m quite sure that will lead you to concur with me that $0.61 per test is relatively inexpensive.

If you’re questioning whether SHERLOCK is generally applicable, I urge you to read Zhang, Collins & coworkers in its entirety to learn more about SHERLOCK’s proven ability to detect and distinguish (1) various bacterial pathogens, (2) single-base cancer mutations in cell-free DNA, and (3) health-related single-nucleotide polymorphisms (SNPs) benchmarked against 23andMe genotyping data as the gold standard of these SNPs.

In an article in Science about SHERLOCK, Harvard University’s George Church (who I’ve proclaimed is The Most Interesting Scientist in the World and is the co-founded of a CRISPR therapeutics company), sums up his reaction in one word: ‘Wow.’ I agree!

The article concludes with Collins saying the Broad is now ‘aggressively exploring’ how to commercialize SHERLOCK and may launch a startup company. But before a diagnostic comes to market, it must pass muster at regulatory agencies such as the U.S. Food and Drug Administration. I’m betting is does.

I welcome your sharing any thoughts or comments about this new CRISPR-based diagnostic method.

Curiously Circular RNA (circRNA) Gets Curiouser

  • circRNA Molecules Have, Oddly, No Beginning or End
  • circRNA Are Now Recognized as Regulators of Gene Expression 
  • A Flurry of New Findings Indicate circRNA Are Also Templates for Synthesis of Proteins Having As Yet Unknown Functions

Electron micrograph of ~3,000-nt circRNA. Taken from Matsumoto et al. PNAS (1990).

About a year ago, my blog titled Curiously Circular RNA pointed out that circular RNA (circRNA) in animals are odd molecules in that, unlike the vast majority of other RNA in animals, circRNA have no structural beginning (5’) or end (3’). This very curious feature has, not surprisingly, stimulated considerable scientific interest in knowing more about these molecules, which were serendipitously discovered some 30 years ago.

Application of next-generation sequencing has revealed that circRNA are actually relatively abundant and evolutionarily conserved, which implicates biological importance rather than inconsequential mistakes during RNA splicing mechanisms. Some circRNA have been shown to have function—circRNA can hybridize to complementary microRNA (miRNA), and thus serve as a kind of ‘sponge’ that influences miRNA-based gene expression. Evidence for circRNA involvement in gene expression continues to grow, as there are now >700 items on “circRNA [and] sponges” in Google Scholar.

Very recently published lines of research (that I’ll outline in what follows) implicate circRNA as coding templates for proteins, which heretofore has been exclusively associated with messenger RNA (mRNA). Current dogma holds that translation of mRNA into protein requires recognition of the 7-methylguanylated (m7G) 5’-cap structure to start ribosome binding, while the 3’-poly(A) tail protects the mRNA molecule from enzymatic degradation and aids in stopping translation, as depicted below.

Taken from Shoemaker & Green Nature Structural & Molecular Biology (2012).

Start and stop structural elements characteristic of mRNA are obviously not present in circRNA, which are literally just circles of RNA. Consequently, finding proteins encoded by circRNA has stirred up controversy about whether such proteins are a new and fundamentally important aspect of genetics or just inconsequential biochemical mistakes.

Translation of circRNA in Fly Head Neurons

Fruit fly. Taken from turbosquid.com

Researchers at The Hebrew University of Jerusalem in Israel in collaboration with a team at Max-Delbruck-Center for Molecular Medicine in Berlin, Germany recently reported in Molecular Cell the first compelling evidence that a subset of circRNA is translated in vivo. The study by Kadener & coworkers was carried out using the common fruit fly (Drosophila melanogaster), which is known to have a number of features that lend to investigations of circRNA: (1) >2,500 fruit fly circular RNAs have been rigorously annotated, (2) these are mostly derive from back-splicing (pictured below) of protein-coding genes, (3) hundreds of which are conserved across multiple Drosophila species, and (4) exhibit commonalities to mammalian circRNA.

Direct back-splicing: a branch point in the 5’ intron attacks the splice donor of the 3’ intron. The 3’ splice donor then completes the back-splice by attacking the 5’ splice acceptor forming a circRNA. Taken from Jeck & Sharpless Nature Biotechnol (2014).

This study by Kadener & coworkers involves a plethora of technically complex experimental procedures and associated jargon, from which I’ve extracted what I believe to be some key points to share. After annotating the Drosophila circRNA open reading frames (cORFs), which, by definition,h have the potential for translation, they searched for evidence of their translation utilizing previously published ribosome footprinting (RFP). This led to identification of 37 circRNAs with at least one specific RFP read, referred to as ribo-circRNAs.

Taken from Jeck & Sharpless Nature Biotechnology (2014)

Several representative ribo-circRNAs were then constructed to each have (pictured below) a metallothionine (MT) promoter and V5 tag to facilitate translation and anti-V5 antibody-based detection of the expected protein after transfection into cells.

To determine whether circRNAs are translated in a more relevant tissue, they set up the RFP methodology in fly heads. A genetic locus named mbl that is known to produce a circRNA (circMbl3) at high abundance was selected for targeted mass spectrometry from a fly head immunoprecipitated MBL. They utilized synthetic peptides to determine characteristic spectra for which to search in the fly head immunoprecipitate and found a consistent and very high confidence hit for a peptide that can only be produced by circMbl3.

Kadener & coworkers extended these fly head findings to mammalian mouse and rat systems, but the most interesting part of this study—in my opinion—dealt with what signals ribosome binding and translation in the absence of the 5’ cap structure present in mRNA. They demonstrated circRNA translation under conditions intended to block normal 5’ cap-dependent translation of mRNA, and concluded that “[untranslated regions] of ribo-circRNAs (cUTRs) allow cap-independent translation [and that] further research is necessary to uncover how these sequences promote translation.”

Remarkably, as you’ll now read, another group of investigators have apparently found how such promotion of circRNA translation can occur.

Translation of circRNA is Driven by N6-Methyladenosine (m6A)

The most abundant modification of RNA in eukaryotes is m6A, which has been recently shown by Li et al. to recruit binding proteins that collectively facilitate the translation of specifically targeted mRNAs—i.e. those “marked” with m6A—through interactions with 40S and 60S ribosome subunit “machinery” that actually carry out translation. Contemporaneously, Yang et al. found that m6A likewise promotes efficient initiation of protein translation from circRNAs in human cells. They discovered that consensus m6A motifs are enriched in circRNAs, and a single m6A site is sufficient to drive translation initiation.

As depicted below, this m6A-driven translation requires initiation factor F4G2 and m6A “reader” YTHDF3. Experiments showed that this translation is enhanced by methyltransferase METTL3/14 and inhibited by demethylase FTO, which enzymatically “add” and “subtract” methyl (Me) groups on specific adenosines (A) in circRNAs, respectively.  It has also been shown to be upregulated upon heat shock, which is a commonly employed method to induce “stress” in cells.

Taken from Yang et al.

Further analyses through polysome profiling, computational prediction and mass spectrometry revealed that m6A-driven translation of circRNAs is widespread, with hundreds of endogenous circRNAs having translation potential. Yang et al. concluded by stating that their “study expands the coding landscape of [the] human transcriptome, and suggests a role of circRNA-derived proteins in cellular responses to environmental stress.”

Zinc Finger Protein in Muscle Cell Development

Finally, and essentially contemporaneously with above mentioned two publications, a third independent investigation reported by Legnini et al. demonstrated selective circRNA downregulation using short-interfering RNAs (siRNAs). These reagents for RNA interference (RNAi) were used in an image-based functional genetic screen of 25 circRNA species, conserved between mouse and human, expression of which are differentially expressed during myogenesis (i.e. formation of muscular tissue) in Duchenne muscular dystrophy myoblasts.

This siRNA/RNAi-based functional analysis provided one interesting case related to zinc finger protein 609 (circ-ZNF609)—a reported miRNA sponge—the phenotype of which could be specifically attributed to the circular form and not to the linear mRNA counterpart. Consistent with the circ-ZNF609 sequence having an ORF, they found that a fraction of circ-ZNF609 RNA is loaded onto polysomes and that, upon puromycin treatment, it shifted to lighter fractions, similar to mRNAs. The coding ability of this circRNA was proved through use of artificial constructs expressing circular tagged transcripts, and by CRISPR/Cas9—the trendy gene editing method about which I’ve already commented multiple times.

Despite all this evidence, Legnini et al. stated that they “have no hints on the molecular activity of the proteins derived from circ-ZNF609 and as to whether they contribute to modulate or control the activity of the counterpart deriving from the linear mRNA.”

In thinking about closing comments about this update in circRNA, I decided to emphasize that investigations in the field of RNA continue to reveal complexities that will require many more years of global attention to unravel and understand. In just the past decade or so we’ve learned about gene regulation by miRNA/siRNA, reclassification of “junk DNA” as encoding a myriad of long noncoding RNA (lncRNA), mRNA regulation by base-modifications, and curious circRNAs that are more than sponges, and likely encode hundreds (if not thousands) of proteins whose functions have yet to be elucidated. Amazing!

What are your thoughts about all of this?

Your comments are welcomed.

Postscript

After writing this blog, Panda et al. at the National Institute on Aging-Intramural Research Program, National Institutes of Health published a paper titled High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs. Here’s a snippet of the abstract which adds to the increasingly curious occurrence of circRNAs that begs, if you will, further research aimed at discovering functions of circRNA-derived proteins.

“Here, we describe a novel method for the isolation of highly pure circRNA populations involving RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion (RPAD), which removes linear RNA to near completion. High-throughput sequencing of RNA prepared using RPAD from human cervical carcinoma HeLa cells and mouse C2C12 myoblasts led to two surprising discoveries: (i) many exonic circRNA (EcircRNA) isoforms share an identical backsplice sequence but have different body sizes and sequences, and (ii) thousands of novel intronic circular RNAs (IcircRNAs) are expressed in cells. In sum, isolating high-purity circRNAs using the RPAD method can enable quantitative and qualitative analyses of circRNA types and sequence composition, paving the way for the elucidation of circRNA functions.”

National ALS Advocacy Day

  • Tuesday May 16, 2017 is National ALS Advocacy Day
  • Mark Your Calendar and Get Involved!
  • Read on to Find Out Why Involvement Matters

Lou Gehrig (1903-1941). Taken from Wikipedia.org

Amyotrophic lateral sclerosis (ALS) is more commonly referred to in North America as “Lou Gehrig disease.” Henry Louis “Lou” Gehrig was a record-setting baseball All-Star for the New York Yankees from 1923 through 1939, when he voluntarily took himself out of the lineup to stunned fans after his play was hampered by ALS. Sadly, Mr. Gehrig died only two years later, which indicates the rapidity of ALS disease progression.

ALS was first found in 1869 by French neurologist Jean-Martin Charcot, but it wasn’t until Lou Gehrig’s affliction that national and international attention was brought to the disease. ALS is now known to be a group of neurological diseases that mainly involve the degeneration of nerve cells (neurons) responsible for controlling voluntary muscle movement, like chewing, walking, breathing and talking. Motor neurons are nerve cells that extend from the brain to the spinal cord and to muscles throughout the body.

Taken from irelandms.com

The disease is relentlessly progressive, meaning the symptoms get continuously worse over time. Both the upper motor neurons in the brain and the lower motor neurons in the spine degenerate or die, and stop sending messages to the muscles. Unable to function, the muscles gradually weaken, start to twitch, and waste away (atrophy). Eventually, the brain loses its ability to initiate and control voluntary movements.

Currently, there is no cure for ALS and no effective treatment to halt, or reverse, the progression of the disease. Most people with ALS die from respiratory failure, usually within 3 to 5 years from when the symptoms first appear. However, about 10 percent of people with ALS survive for 10 or more years.

It is generally estimated there are over 30,000 people living with ALS in the United States at any given time, and that the number worldwide is around 450,000. Someone is diagnosed with ALS every 90 minutes. The life-time incident rate for an average person is often estimated at between 1-in-400 to 1-in-600 people—an incidence rate comparable to that for multiple sclerosis.

Who Gets ALS and Why?

Following are some reliable facts that I selected from an authoritative NIH website for ALS:

  • ALS affects people of all races and ethnic backgrounds.
  • Caucasians and non-Hispanics are most likely to develop the disease.
  • Although the disease can strike at any age, symptoms most commonly develop between the ages of 55 and 75.
  • Men are slightly more likely than women to develop ALS. However, the difference between men and women disappears with aging.
  • Military veterans are about 1.5 to 2 times more likely to develop ALS, and is recognized as a service-connected disease by the U.S. Department of Veterans Affairs.
  • 90% of ALS cases are considered sporadic, i.e. the disease seems to occur at random.
  • 10% of ALS cases are familial, i.e. an individual inherits the disease from his or her parents.
  • Mutations in more than a dozen genes have been found to cause familial ALS

How is ALS treated?

Riluzole. Taken from healthtap.com

No cure has yet been found for ALS. However, there are treatments available that can help control symptoms, prevent unnecessary complications, and make living with the disease easier. In 1995, the FDA approved riluzole (Rilutek), the only disease-modifying drug to date for ALS. Riluzole has multiple neural mechanisms of action, and is believed to reduce damage to motor neurons by decreasing levels of glutamate, which transports messages between nerve cells and motor neurons. Unfortunately, clinical trials in people with ALS showed that riluzole prolongs survival by only a few months.

BrainStorm Cell Therapeutics & NurOwn®

In researching clinical trials for hopefully much more effective therapies for ALS, I came across a company in Israel named BrainStorm Cell Therapeutics Inc. (BCTI) that offers a form of stem cell therapy for ALS that appears to be quite promising. Following is a short synopsis of what I found.

Space-filling model of BDNF. Taken from Wikipedia.org

BCLI has developed a patented stem cell-based technology that delivers a growth factor that can help neurons live longer at or near the site of injury or damage. More specifically, a mesenchymal stem cell isolated from an ALS patient is grown in a cell culture under certain conditions to produce a differentiated phenotype that secretes brain-derived neurotrophic factor (BDNF) at a level at least five-times greater than normal. The term “factor” is generic in biomedical parlance, and in the case of BDNF refers to a protein pictured to the right.

After these “super secreting” cells are obtained ex vivo, they are reintroduced (aka implanted) into the same ALS patient (i.e. an autologous transplant; see schematic diagram) wherein BDNF acts on neurons of the central nervous system and the peripheral nervous system, helping to support the survival of existing neurons, and encourage the growth and differentiation of new neurons and synapses.

Taken from Byrne JA. Overcoming clinical hurdles for autologous pluripotent stem cell-based therapies. OA Stem Cells 2013 Sep 01;1(1):3.

Details for this cell-harvesting, ex vivo differentiation, selection, and implantation can be read in this 2014 article by BCTI in Clinical Translation Medicine. BCTI has registered these differentiated BDNF-secreting cells as NurOwn®, which I assume is intended to sound bit like “your own”—to reflect the autologous nature of the transplant—and be a linguistic blend of neuron and own. In any case, the important point is that safety and efficacy have been reported in a recently published Phase 1/2 and 2a open-label, proof-of-concept studies of patients with ALS at the Hadassah Medical Center in Jerusalem, Israel.

In the Phase 1/2 part of the trial, 6 patients with early-stage ALS were injected intramuscularly (IM) and 6 patients with more advanced disease were transplanted intrathecally (IT), i.e. via the spinal cord. In the Phase 2a dose-escalating study, 14 patients with early-stage ALS received a combined IM and IT transplantation of autologous BDNF-secreting cells. Interested readers can consult this publication for details, but the bottom line, if you will, was that the possible clinical benefits would be hopefully confirmed in an upcoming clinical trial.

My further research on this led to a February 2017 press release by BCTI announcing that City of Hope’s Center for Biomedicine and Genetics (CBG) in Duarte, California will produce clinical supplies of NurOwn® adult stem cells for the BCTI’s planned randomized, double-blind, multi-dose Phase 3 clinical study in patients with ALS. It added that CBG is expected to support all U.S. medical centers that will be participating in the Phase 3 trial.

A second February 2017 press release by BCTI announced an agreement with Centre for Commercialization of Regenerative Medicine (CCRM) in Toronto, Canada to support the market authorization request for NurOwn® and explore the opportunity to access Health Canada’s early access pathway for treatment of patients with ALS as early as 2018.

Other ALS Clinical Studies

My “go to” authoritative source of reliable information about clinical trials is NIH’s ClinicalTrials.gov website that has an updated database that can be searched and filtered in many ways. When I searched for ALS clinical trials that were recruiting patients, I was heartened to find over 180 studies that can be perused via this link. For each study, there is information about purpose, study design and measures, eligibility, contacts and locations.

Incidentally, as regular readers of my blog will know, modified mRNA (mod mRNA) therapeutics is a relatively new and very promising modality for treating diseases that respond to providing or supplementing proteins. Given that DNA vectors encoding BDNF mRNA are readily available, I’m hoping that a mod mRNA for BDNF will soon be investigated as yet another avenue of treatment for ALS.

Advocate for ALS!

Taken from alsa.org

The ALS Association (ALSA) has the stated mission “to discover treatments and a cure for ALS, and to serve, advocate for, and empower people affected by ALS to live their lives to the fullest.” ALSA’s website offers various ways for any individual to become an advocate for ALS, such as becoming informed and donating much needed money or time, participating in the Walk to Defeat ALS® that draws people of all ages and athletic abilities together (see picture) to honor the courageous souls who are affected by ALS, to remember those who have passed, and to show support for the cause.

One of my long-time friends has recently been diagnosed with ALS, which in part led me to research this blog to help inform him, and led me to find a Walk to Defeat ALS® in which to participate. I encourage you to do advocate for ALS in whatever way you wish and are able.

As usual, your comments are welcomed.

DNA Day 2017

  • There are Now Millions of DNA-Related Publications
  • Some of the Top 5 Cited Papers on DNA Will Surprise You
  • You Probably Won’t Guess Top 5 Most Frequently Cited

Deciding what to post here in recognition of DNA Day 2017 was just as challenging as it has been in past years, primarily because there’s so many different perspectives from which to choose. After much mulling, and several abandoned approaches, I settled on featuring DNA publications that have received the most citations, as an objective metric—not just my subjective opinions about topics I think are significant or otherwise interesting.

Before getting to the numbers of DNA-related papers and some of the most cited papers, here’s a quick recap of what was posted here in the past, starting with the inaugural blog four years ago:

2013—60th Anniversary of the Discovery of DNA’s Double Helix Structure

2014—My Top 3 “Likes” for DNA Day

2015—Celebrating Click Chemistry in Honor of DNA Day

2016—DNA Dreams Do Come True!

Explosive Growth of DNA Publications

Regular readers of my blogs will know that I frequently use the NIH PubMed database of scientific articles to find publications by searching keywords, phrases, or authors. A convenient feature of these searches is providing “results per year” that can be exported into Excel for various purposes. Some preliminary searches indicated that DNA-related articles can be indexed by either DNA or PCR, or cloning, or other terms among which sequencing was notable. The majority, however, were indexed as either DNA or PCR, which together gave nearly 1.7 million items—an astounding number. This number is even much greater since PubMed excludes some important chemistry journals, as well as patents.

Diving deeper into these numbers, I thought it helpful to look at the publication volumes and rates for DNA, sequencing DNA, and PCR through 2015 starting from 1953, 1977, and 1986, respectively. These respective dates correspond to seminar publications by Watson & Crick, Maxam & Gilbert, and Mullis & coworkers. The results shown in the following graph attest to my often stated “power of PCR” as premier method in nucleic acid research, which we’ll see again below in another numerical context.

Top 5 Cited Papers

During my perusal of the above literature in PubMed generally related to DNA, I thought it would be interesting to find, and share here, which specific papers have the distinction of being most frequently cited. Citations are not available in PubMed, but are compiled in Google Scholar, which led me to these Top 5 that are listed from first to fifth.

Frederick Sanger (1918-2013) Taken from newscientist.com

  1. DNA sequencing with chain-terminating inhibitors

Frederick Sanger, the eponymous father of the “Sanger sequencing” method published in 1977, received the 1980 Nobel Prize in chemistry for this contribution. He also received the 1958 Nobel Prize in chemistry for sequencing insulin, and is the only person to win two Nobel Prizes in chemistry. Uber-famous DNA expert Craig Venter is quoted as saying that ‘Fred Sanger was one of the most important scientists of the 20th century,’ [who] ‘twice changed the direction of the scientific world.’

  1. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method

Kenneth J. Livak, PhD
Taken from archive.sciencewatch.com

The most commonly used method to analyze data from real-time, quantitative PCR (RT-qPCR) experiments is relative quantification, which relates the PCR signal of the transcript of interest to that of a control sample such as an

untreated control. The derivation, assumptions, and applications of this method were published in 2001 by Livak & Schmittgen. I overlapped with Ken Livak at Applied Biosystems, which pioneered commercilaization of RT-qPCR reagents and instrumentation at the time. He is currently Senior Scientific Fellow at Fluidigm Corp.

Sir Edwin M. Southern Taken from ogt.co.uk

3. Detection of specific sequences among DNA fragments separated by gel electrophoresis

Sir Edwin Mellor Southern, FRS, the eponymous father of “Southern blotting” DNA fragments from agarose gels to cellulose nitrate filters published in 1975, is a Lasker Award-winning molecular biologist, Emeritus Professor of Biochemistry at the University of Oxford and a fellow of Trinity College. He is also Founder and Chief Scientific Advisor of Oxford Gene Technology.

  1. Prof. Bert Vogelstein, MD
    Taken from hhmi.org

    A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity

This paper by Feinberg & Vogelstein published in 1983 describes how to conveniently radiolabel DNA restriction endonuclease fragments to high specific activity using the large fragment of DNA polymerase I and random oligonucleotides as primers. These “oligolabeled” DNA fragments serve as efficient probes in filter hybridization experiments. His group pioneered the idea that somatic mutations represent uniquely specific biomarkers for cancer patients, leading to the first FDA-approved DNA mutation-based screening tests, and now “liquid biopsies” that evaluate blood samples to obtain information about underlying tumors and their responses to therapy (an area that I’ve touted in previous blogs). A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 109 dpm/μg of DNA can be obtained using relatively small amounts of precursor. These “oligolabeled” DNA fragments serve as efficient probes in filter hybridization experiments.

  1. Kary B. Mullis, PHD
    Taken from TED.com

    Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase

In 1988, Kary B. Mullis and coworkers (then at Cetus Corp.) published in venerable Science a method using oligonucleotide primers and thermostable DNA polymerase from Thermus aquaticus to amplify genomic DNA segments up to 2000 base pairs to detect a target DNA molecule present only once in a sample of 105 cells. Since that time, polymerase chain reaction (PCR)-related technology has evolved to now routinely enable a variety of single-cell analyses of DNA or RNA. Dr. Mullis received the 1993 Nobel Prize in chemistry for his 1983 invention of PCR, which his website says ‘is hailed as one of the monumental scientific techniques of the twentieth century.’

Top 5 Papers by Citation Frequency

While writing the above section, it occurred to me that ranking these five publications by total number of citations-to-date in Google Scholar doesn’t account for differences in the number of years between the year of publication and now. I did the math to calculate the average citation frequency per year, and here’s the totally surprising—to me—result: relative gene expression methodology published by Livak & Schmittgen is by far the most frequently cited of the Top 5, according to this way of ranking:

  1. 2001, relative gene expression, Cited by 69560 = 4,637 avg. citations per year
  2. 1977, Sanger sequencing, Cited by 32662 = 1,701
  3. 1975, Southern blotting, Cited by 21201 = 796
  4. 1988, PCR, Cited by 18785 = 671
  5. 1983, oligolabeled DNA, Cited by 21200 = 642

I should point out that, as transformative methods such as these gradually become widely recognized as “standard procedures,” researchers tend to feel it unnecessary to include a reference to the orignal publication. Consequenly, citation frequency decreases with time even though cummulative usage increases. In other words, 25 years from now average citations per year for relative gene expression will have likely decreased, and be surpassed by a new “method of the decade,” so to speak.

Prediction for the Future

This line of reasoning leads me to close with some speculation about what DNA-related technique might emerge as the next “method of the decade” that tops the above ranking by citation frequency.

My guess is that it will be Multiplex genome engineering using CRISPR/Cas systems by Zhang & coworkers that has been cited by 4145 at the time I’m writing this piece, only four years from its publication in venerable Science in 2013. Some of my blogs have already commented on various aspects of CRISPR/Cas9, which is among genome editing tools offered by TriLink.

As usual, your comments are welcomed.

Autism Awareness Month – April 2017

  • Sequencing for Diagnosis of Autism Holds Promise
  • Several Genetic-Risk Testing Procedures are Available
  • More Than 40 Autism Publications Using TriLink Products

The first National Autism Awareness Month was declared by the Autism Society in April 1970 with the aim of educating the public about autism. Autism is a complex mental condition and developmental disability, characterized by difficulties in the way a person communicates and interacts with other people. Autism can be present from birth or form during early childhood, typically within the first three years. Autism is a lifelong developmental disability with no single known cause.

The puzzle pattern of this ribbon reflects the complexity of autism, while the colors and shapes represent the diversity of people and families living with this spectrum of disorders. Taken from drdiane.com

People with autism are classed as having Autism Spectrum Disorder (ASD) and the terms autism and ASD are often used interchangeably. The term “spectrum” refers to the wide range of symptoms, skills, and levels of disability in functioning that can occur in people with ASD, which includes Asperger syndrome. Some children and adults with ASD are fully able to perform all activities of daily living while others require substantial support to perform basic activities. ASD occurs in every racial and ethnic group, and across all socioeconomic levels. However, boys are significantly more likely to develop ASD than girls. The latest analysis from the U. S. Centers for Disease Control and Prevention (CDC) estimates that 1 in 68 children has ASD.

Taken from myaspergerschild.com

According to the CDC, diagnosing ASD can be difficult, since there is no medical test, like a blood test, to diagnose the disorders. Doctors look at the child’s behavior and development to make a diagnosis.” More details from the CDC are provided at this link.

Notwithstanding this current difficulty for diagnosis of ASD, research has led to continuing progress toward possible blood tests for ASD, which is the focus of this blog and supplements an earlier posting here on treating autism with a broccoli nutraceutical.

ASD and Exome Sequencing

My Google Scholar search for “autism and sequencing” led to a mindboggling list of more than 47,000 items! When ordered by relevance rather than date of publication, two publications were each cited ~1,000-times following “back-to-back” appearance in venerable Nature magazine in 2012. This computes to a combined average of ~400 citations per year, or a fraction more than one citation per day on average, which to me signals significant attention by the ASD research community and thus worth commenting on herein.

Sanders et al., in the first of these two widely cited studies, carried out exome sequencing in 238 families wherein each pair of parents was unaffected by ASD but had a child who was affected (aka proband), and in 200 of these families there was an unaffected sibling. This study design feature is important in view of the widely held idea that complex personality traits are derived by a combination of “nature and nurture,” i.e. genetics inherited from parents and that which is learned or otherwise acquired by familial and all external events.

Before synopsizing what was found, I should note that germline single-base mutations spontaneously arise during mitosis in every generation, and are termed de novo single nucleotide variants (SNVs). Identifying SNVs remained refractory to analysis at the whole genome or exon level until the advent of next-generation sequencing (NGS) technologies.

Sanders et al. found that the total number of non-synonymous (i.e. changes in the amino acid sequence of proteins) de novo SNVs—particularly highly disruptive nonsense and splice-site de novo mutations—are associated with ASD. They concluded that their results “substantially clarify the genomic architecture of ASD, demonstrate significant association of three genes—SCN2A, KATNAL2 and CHD8—and predict that approximately 25–50 additional ASD-risk genes will be identified as sequencing [more] families is completed.”

Neale et al., in the second widely cited study, likewise conducted exome sequencing but on only 175 ASD probands and their parents. Nevertheless, they found that the proteins encoded by genes that harbored de novo non-synonymous or nonsense mutations showed a higher degree of connectivity among themselves and with previous ASD genes as indexed by protein-protein interaction screens. They concluded that their results “support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5- to 20-fold,” but did acknowledge the strong evidence reported by Sanders et al. for individual genes as risk factors.

ASD Genetic-Risk Testing

The American Academy of Pediatrics (AAP) in 2013 issued a statement on ethical and policy issues for genetic screening of children for ASD that was prompted in part due to then recent progress by IntegraGen—a small French genomics company—on development of a gene test that uses a cheek swab to screen infants and toddlers for 65 genetic markers associated with autism. Highlights of the AAP’s statement include:

  • Genetic screening can be particularly useful for diagnosing older babies and children with developmental disorders such as autism.
  • Genetic screening should be made available for all newborns. However, parents should have the right to refuse screening after being informed of the benefits and risks.
  • The decision to offer testing or screening should be based primarily on the best interest of the child.

Taken from autismspeaks.org

By way of an update, I’m pleased to add that in a 2015 press release by IntegraGen it was announced that its ARISk® Test became the first test marketed in the U. S. to assess the risk of autism spectrum disorder in children. Among the following IntegraGen statements about the ARISk® Test, I think it’s most important to note the caveats I’ve bolded for emphasis:

  • The test does not confirm or rule out a diagnosis of ASD for the child tested.
  • The test is intended to be used together with a clinical evaluation and other developmental screening tools.
  • Intended for children with early signs of developmental delay or ASD and in children who have older siblings previously diagnosed with an autism spectrum disorder.
  • A genetic score, based on the total number of genetic markers associated with autism identified, is used to estimate the child’s risk of developing ASD.
  • Intended for use for children 48 months and younger. The ARISk® Test is not available for prenatal testing.

Taken from integragen.com

More recently, Courtagen—cofounded by my former Life Technologies colleague Kevin McKernan (coinventor of SOLiD® NGS)—has commercialized its sequencing analyses for ASD and other neurodevelopmental conditions. According to a Courtagen posting, “[i]n the absence of a known single-gene disorder, ASD likely involves a complex combination of both genetic and environmental factors that influence early brain development. Multi-gene panels, such as Courtagen’s devSEEK® panels, provide clinicians with information on a number of genes commonly associated with ASD and autistic features. Clinicians can then use information from multi-gene panels to tailor treatments that meet the patient’s unique genotype and symptoms.”

Some interesting—to me—logistical and operational information about devSEEK® (237 genes) is as follows:

  • Turn-around time for results is 4-6 weeks.
  • DNA for sequencing is extracted from a single saliva sample. No blood draw or muscle biopsy required; however, blood and muscle tissue are accepted.
  • Courtagen works with patients, physicians, and insurance carriers to pre-approve each test. Courtagen will bill the insurance company and is willing to handle an appeal process as needed.
  • A secure physician online portal is available for ordering genetic tests and accessing patient reports when completed. Genetic counselors are available to address questions regarding Courtagen test results.

ASD Research and TriLink

While mulling over how to conclude this Autism Awareness Month blog featuring genetic testing for ASD, I wondered about TriLink’s role in advancing autism research by virtue of its various nucleic acid-related products being used for autism investigations. I was pleased and proud to find more than 40 items by searching Google Scholar for articles with the words “autism and TriLink.”

Perusal of these items revealed that the most cited (450-times) report was a 2012 publication in highly regarded Cell titled MeCP2 Binds to 5hmC Enriched within Active Genes and Accessible Chromatin in the Nervous System, which used TriLink 5-methyl-2′-deoxycytidine-5′-triphosphate (5m-dCTP). Given the apparent significance of this publication, I won’t try to give a short, simplified synopsis but rather quote the following part of the authors’ summary:

“We report that 5hmC [5-hydroxymethylcytosine] is enriched in active genes and that, surprisingly, strong depletion of 5mC [5-methylcytosine] is observed over these regions. The contribution of these epigenetic marks to gene expression depends critically on cell type. We identify methyl-CpG-binding protein 2 (MeCP2) as the major 5hmC-binding protein in the brain and demonstrate that MeCP2 binds 5hmC- and 5mC-containing DNA with similar high affinities. The Rett-syndrome-causing mutation R133C preferentially inhibits 5hmC binding. These findings support a model in which 5hmC and MeCP2 constitute a cell-specific epigenetic mechanism for regulation of chromatin structure and gene expression.”

I also noted a 2016 Cutting-Edge Review in Arteriosclerosis, Thrombosis, and Vascular Biology titled A CRISPR Path to Engineering New Genetic Mouse Models. These investigators utilized TriLink Cas9 mRNA for gene editing analogous to that reported by others for CRISPR/Cas9-mediated knockout of the autism gene CHD8 (see above). This led to transcriptomic profiling showing that CHD8 regulates multiple genes implicated in ASD pathogenesis and genes associated with brain volume.

In conclusion, I must say that I learned much new information about autism while researching this blog, which I hope you found informative as well as interesting. If so, I have achieved my goal of either increasing or reaffirming your awareness of autism, and the availability of genetic risk-assessment tests.

As usual, your comments here are welcomed.

Postscript

Recently, a team of academic researchers in Arizona made headlines with their publication in Microbiome reporting ties between autism symptoms and the composition and diversity of a person’s gut microbes, aka “gut microbiome,” about which I’ve commented on in several previous blogs.

The participants, who were 18 children with ASD (ages 7–16 years), underwent a 10-week treatment program involving antibiotics, a bowel cleanse, and daily fecal microbial transplants over 8 weeks. Remarkably, the new therapy seemed to provide some long-term benefits, including an 80% improvement of gastrointestinal symptoms associated with ASDs and roughly a 20% – 25% improvement in autism behaviors, including improved social skills and better sleep habits.

Click here for a simplified, educational video on this work by the principal investigator, Prof. James B. Adams at Arizona State University.

I should emphasize that this is a very small study, and much more research will be needed to verify and firmly establish possible benefits and risks. Interested readers should contact Prof. Adams regarding any questions they might have.

Finding Frankfurter Fraud Featuring Famously Familiar PCR

  • While Thousands of PCR-based Tests for Food Authentication Exist, Commercial Adoption is Lax
  • PCR Tests for Halal Frankfurter Products Reported by Malaysian Team
  • PCR-enabled Next Generation Sequencing of USA Frankfurters Exposes Extensive Mislabeling and Adulteration

Regular readers of this blog will know that (a) I favor alliterations, (b) frequently feature PCR-based topics, and (c) I am fond of food facts involving nucleic acids, all of which are found in the title of this posting. While writing style and food are both a matter of taste, so to speak, it’s almost impossible to comment on nucleic acids without involving PCR, in one way or another, as PCR is—in my opinion—the most widely used and important method in molecular biology.

Having said this, and knowing that this summer alone Americans will likely consume an estimated 7 billion (!) hot dogs, (a.k.a. frankfurters or wieners), I thought it apropos to now feature finding frankfurter fraud by PCR. But before getting to that, I thought it’s worth commenting first on a frankfurter vs. wiener vs. hot dog and other “meaty” definitions to get us “linked” up—please pardon the puns, another of my penchants.

Frankfurter, Wiener, Hot Dog Lexicon

Frankfurter sausages and sauerkraut. Taken from tripadvisor.com

While it’s a fact that a resident of Frankfurt, Germany is properly called a Frankfurter, one of Frankfurt’s pork sausage specialties is also called a frankfurter—but spelled with lower case f—and is short for Frankfurter Würstchen, which go back to the 13th century. By the same token, wiener refers to a pork and beef sausage specialty introduced in the 18th century in Vienna, Austria—a city called Wien in German, and hence the word wiener.

Frankfurters and wieners look very similar, with the main culinary distinction being absence of serving with the bun, which by contrast is characteristic of a hot dog. Readers interested in the origin of the hot dog’s name and bun usage will learn at this link—pun intended—that there are various and widely different claims for this name and usage.

Hot dogs in buns. Taken from clowns4kids.com

Dog Factory, a short film by uber-famous Thomas Edison in 1904 poked fun at what went into hot dogs. Taken from wikipedia.org

In one such claim, the term dog is said to have been linked—there I go again—to sausages made from dog meat, as popularized in an old spoof by Thomas Edison (!) pictured below. This issue of what meat(s) hot dogs and similar sausages contain now segues into finding frankfurter fraud using PCR.

Finding Food Fraud by PCR

Before getting to the meat of this matter (oops!) involving frankfurters, I thought it would be informative to provide some larger perspective on finding food fraud, generally, utilizing the power of PCR for specific detection of nucleic acids that are characteristic of a given species of any food whether it be meat, fish, vegetable, etc. My search of Google Scholar for articles with all of the terms “food, fraud, and PCR” gave ~3,900 items. Here are some selected samplings, the first of which was taken from several I found that used TriLink products—hooray!

  • US Food & Drug Administration researchers employed species-specific primers from TriLink for multiplex real-time PCR analysis of salmon and trout species in a range of 80 commercial products in North America. 4 instances (5%) of fraud were found.
  • By contrast, a whopping 40% of commercial pet food products tested by PCR for the presence of eight meat species (bovine, caprine, ovine, chicken, goose, turkey, porcine, and equine) were found to be potentially mislabeled, according to a study by academic researchers.
  • Saffron produced from dried stigma of Crocus sativus is considered to be the most expensive food spice in the world, as ~200,000 (!) flowers must be carefully hand-picked (!) to produce only 1 kg of spice. Combating saffron fraud with PCR has already led to ~150 publications (!).
  • PCR-based food authentication to screen for possible allergens or GMOs is important for prevention of potentially life-threatening food contamination or alleviating consumer perceptions—or perhaps misperceptions, as I’ve commented on previously.

Finding Frankfurter Fraud by PCR

As for finding frankfurter fraud by PCR, the aforementioned ~3,900 items from my Google Search of food, fraud, and PCR was sub-searched for frankfurter, which led to only 3 reports titled as follows:

The third item, which is by a team of Malaysian researchers and is the most recent, offers some interesting introductory perspectives on strict religious, cultural, or geographical restrictions over the consumption of certain meats in the context of commercial frankfurters.

For example, pork is totally unacceptable to Malaysia’s large Muslim population, as well as Jewish and certain Christian denominations. On the other hand, Egyptians prefer buffalo because of their cultural preferences, while some Indians and Europeans avoid beef because of religious requirements and the fear of bovine spongiform encephalopathy (aka “Mad Cow Disease”—click here for an FDA update), respectively.

In this Malaysian study, 100% beef, buffalo, or pork frankfurters were prepared as models, and then fully heat-processed in the laboratory to simulate conventional manufacturing procedures. Additional beef, buffalo, or pork frankfurter models were deliberately contaminated by “spiking” in 1%, 0.5%, or 0.1% of buffalo and pork, beef and pork, and beef and buffalo meat, respectively. PCR was then performed using species-specific primer pairs for two genes (cytochrome b and NADH dehydrogenase subunit 5) for cross-validation. Twenty different halal-branded (i.e. pork-free) “beef” frankfurters from Malaysian markets were tested. While no pork was detected in any of the tested “beef” frankfurters, they were all beef- and buffalo-positive, thus revealing that all of the investigated Malaysian commercial “beef” products were buffalo-adulterated.

Halal Certification by PCR

The above mentioned concern for non-pork halal-assurance piqued my interest as to the extent of PCR usage for halal-related certification, and my subsequent Google Scholar search for “halal, certification, PCR” gave nearly 350 items. This relatively large number of reports signals quite widespread adoption of PCR. I encourage those interested to peruse these items later, but will mention here that the following article is the most cited—close to 100 citations as of February 2017—Identification of pork derivatives in food products by species-specific polymerase chain reaction (PCR) for halal verification

In closing, I think it’s worth noting in the context of halal certification—and increasingly popular “democratization” of technology, about which I’ve previously offered comments—a French start-up company now offers an antibody-based “dip stick” kit (Halal Test) for anyone to use for pork-free halal-assurance at home or even when eating out. Amazing.

Taken with copy write permission from French duo launch HalalTest: ‘We want to democratize analysis’ by Rachel Arthur+Rachel ARTHUR, 05-Nov-2014

Hot Dog! — There’s Now an NGS Food Authentication Service Company

Frankly—pun-to-be intended—I don’t know how “hot dog!” became an exclamatory phrase for good news. It applies, however, to the fact that Clear Labs Inc. (a 2014 start-up in Palo Alto, California) has proven that several issues of concern for consumers of hot dogs can be successfully addressed by PCR/NGS methods.

In a Clear Labs’ poster abstract for the 2016 International Association of Food Protection meeting, results were reported for a study of 345 hot dog products sold by national brands to compare product label information and ingredient lists with the results of NGS analyses. Following DNA extraction, universally accepted regions for animals, plants, and bacteria were PCR-amplified for NGS, which revealed that ~15% of these products had ingredient substitution, unexpected ingredients, or hygienic issues. In addition, 10% of all products labelled as vegetarian contained detectable levels of meat DNA. Vegetarian products also accounted for 67% of hygienic issues, such as human DNA.

At the risk of overly generalizing these findings, I believe that they probably reflect very widespread issues in the food industry, which we as consumers are virtually helpless to deal with, and can only hope that US FDA regulations begin to mandate PCR-based food certification.

Having said this, I didn’t want to end with a “downer.” Therefore, I’ll conclude with some hopefully fun facts about hot dogs, which—if you’re wondering—can be made from beef, pork, turkey, chicken, or a combination (but must be labeled as such, according to FDA information worth reading later).

Fun Frankfurter (aka Hot Dog) Facts

Just for fun, I calculated that the 7 billion frankfurters to-be-consumed by Americans this summer would stretch 56,818 miles (assuming each one were 6 inches long), which would wrap around Earth’s equator 2.3 times if placed end-to-end!

Some other fancinating frankfurter feats:

  • Taken from wikipedia.org

    The world’s longest hot dog was 197 feet and was prepared by Shizuoka Meat Producers and the All-Japan Bread Association for the latter’s 50th anniversary celebration in 2006 at the Akasaka Prince Hotel:

  • The world’s most expensive hot dog is the “California Capitol City Dawg”, served at Capitol Dawg in Sacramento, California and cost $145.49. Proceeds from the sale of each 18-inch long, 3-pound super “Dawg” are donated to the Shriners Hospitals for Children.
  • The annual Nathan’s Hot Dog Eating Contest is held on Independence Day at Nathan’s Famous original, and best-known restaurant in Coney Island, a neighborhood of Brooklyn, New York City. The current champion is Joey “Jaws” Chestnut, who ate a stomach-busting 70 (!) hot dogs and buns in only 10 minutes (!) at the 2016 championship.

Taken from elitedaily.com

As usual, your comments here are welcomed.

Evolving Polymerases to Do the Impossible

  • Polymerases Aren’t What They Used to Be! 
  • Scripps Team Evolves Polymerases That Read and Write With 2’-O-Methyl Ribonucleotides
  • Key Reagents for Romesberg’s “Molecular Moonshots” Are Supplied by TriLink BioTechnologies

Long-time devotees of these posts will likely remember a blog several years ago about Prof. Floyd Romesberg at the Department of Chemistry, The Scripps Research Institute who achieved a seemingly impossible feat. Namely, designing a new pair of complementary bases such that DNA replicating in E. coli would be comprised of six bases, thereby creating a six-base genetic code that is expanded from Nature’s four-base code.

Floyd E. Romesberg. Taken from utsandiego.com

More recently, Romesberg has cleverly outfoxed Nature once again, this time by evolving nucleic acid polymerases into mutant polymerases that can do what heretofore seemed impossible. He and his research team’s publication (Chen et al.) is a tour de force of experimental methodology that is not easily read, and is even harder to simply summarize in a short space like this blog. Consequently, I’ll first tell you what was accomplished, then give a short synopsis of principal new methodology, and close by commenting on the significance of this fascinating work.

Doing the Impossible

Romesberg’s lab successfully achieved what I think of as “multiple molecular moonshots,” wherein a Taq polymerase (which normally reads and writes DNA during PCR), was evolved by novel selection (SELEX) methods into mutant polymerases that are able to transcribe DNA into 2’-O-methyl (2’-OMe) RNA, and reverse transcribe 2’-OMe RNA into DNA for PCR/sequencing.

As depicted below, this was exemplified using a 60-mer DNA template and 18-mer 2’-OMe RNA primer to produce a fully-modified 48-mer 2’-OMe RNA by means of an evolved mPol and all four A, G, C and U 2’-OMe NTPs, which I’m proud to say were bought from TriLink BioTechnologies! This type of molecular evolution of a polymerase has no precedent.

DNA template   5’ ————————————- 3’

RNA primer                                 ←←← 3’ xxxxxx 5’

mPol ↓ 2’-OMe NTPs

Determining the fidelity of this seemingly impossible molecular transformation was addressed by achieving a feat of comparable impossibility! As depicted below, the aforementioned 48-mer 2’-OMe RNA product was hybridized to a DNA primer for reverse transcription into a 48-mer complementary DNA (cDNA) strand, using an evolved mPol, together with all four A, G, C and T unmodified dNTPS, which were also purchased from TriLink. This unprecedented conversion of 2’-OMe RNA into cDNA was followed by conventional PCR/sequencing, the results of which demonstrated relatively high fidelity.

2’-OMe template   5’ xxxxxxxxxxxxxxxxxxxxxxxxxxx 3’

DNA primer                                           ←←← 3’ —— 5’

mPol ↓ dNTPs

cDNA                        3’ ————————————– 5’

How They Did It

In the selection cycle shown below, (1) phage-display libraries were used to expose individual polymerases (Pol) on E. coli. cells in proximity to chemically attached primer/template complexes of interest, which are mixed with natural or modified triphosphates including biotin (green; B)-labelled UTP to extend the primer. (2) Phage that display active mutant polymerases (mPols) are isolated with streptavidin (SA) beads. After washing to remove nonspecific binders, phage cleaved from the beads are used to re-infect E. coli. (3) Heat-treated lysates of E. coli that express the recovered mPols are next subjected to plate-based screening using 96-well plates coated with primer/template complex and extension buffer that contained natural or modified triphosphates and B-UTP, incorporation of which is chromogenically detected. (4) Mutants that give rise to the most activity are selected for individual gel-based analysis, from which (5) promising candidates are selected for further diversification (e.g., by gene shuffling, as depicted) and then subjected to additional rounds of evolution.

Taken from Chen et al. Nature Chemistry (2017)

What is the Significance

In a previous blog, I’ve commented on increasing interest in the utility of aptamers, which are oligonucleotides that can specifically bind small molecules or motifs in proteins, and thus be used to build electronic sensors or studied as potential therapeutic agents rivaling antibodies. Therapeutic aptamers, like antisense oligonucleotides, require incorporation of chemical modifications to impart stability toward nucleases in blood or cellular targets.

Burmeister et al. have previously reported methods for mPol transcription of a DNA template into a fully modified, nuclease-resistant 23-mer 2’-OMe RNA aptamer—also using TriLink’s 2’-OMe NTPs! However, they encountered considerable experimental difficulties in generating this therapeutically promising 23-mer against vascular endothelial growth factor. These technical issues have now been surmounted by the mPol-evolution approaches in the present work by Romesberg’s team, which enabled improved access to longer 2’-OMe RNA aptamers with reasonable efficiency and fidelity.

Moreover, the present study is the first to evolve an mPol for reverse transcription of fully modified 2’-OMe RNA into DNA, which can then be amplified by PCR and/or sequenced, thereby opening the door for a variety of new analytical methods. Most importantly, the molecular mechanism by which these remarkable mPol activities was evolved, namely, the stabilization of an interaction between the “thumb and fingers domains,” may be general and thus useful for the optimization of other Pols. In that case, we can look forward to further advances in evolving other Pols to do the impossible—hopefully using modified nucleotide triphosphates from TriLink!

As usual, your comments are welcome.

CRISPR-Mediated Interference (CRISPRi) of Long Non-Coding RNA (lncRNA)

  • More Methodology from CRISPR Mania
  • lncRNA Function Blocked by CRISPRi
  • Mysteries of lncRNA Can Now be Deciphered by CRISPRi

This blog is about yet another example of a powerful new methodology spawned by intense scientific interest in using CRISPR-related technologies. This near mania for all things CRISPR is reflected by there being ~5,000 (!) publications already in PubMed only ~5 years after seminal papers appeared.

I chose the present blog topic because it involves use of CRISPR for genome-wide identification of functional long non-coding RNA (lncRNA) in human cells. In an earlier blog about lncRNA, which are now recognized to be regulators of gene expression encoded by what was originally defined as “junk” DNA, it was pointed out that it is inherently difficult to experimentally identify such regulation by lncRNA. Thanks to CRISPR this task is now much less daunting as you’ll learn below, following a couple of introductory sections to set the stage.

Repurposing CRISPR/Cas9 Using “Dead” Cas9

Qi et al. very cleverly—at least to me—recognized that the CRISPR/Cas9 system could be repurposed as an RNA-guided platform for sequence-specific control of gene expression by finding a catalytically inactive mutant Cas9 protein that lacked exonuclease (i.e. cutting) activity of wild-type Cas9, and instead blocked transcription by RNA polymerase (RNAP), as depicted below. These researchers coined the overall process as “CRISPR interference” (CRISPRi) and loosely referred to such a mutant Cas9 as “dead” Cas9 (dCas9).

Taken from Qi et al.

Interested readers are encouraged to consult this publication by Qi et al. to fully appreciate the extensive amount of work that went into translating the above concept into practice, and supporting the proposed mechanism of action. In my opinion, it’s a tour de force example of applying hypothesis-driven, state-of-the art molecular biology to devise a new method—in this case specifically blocking transcription of a DNA region using CRISPRi in conjunction with target-specific short guide RNA (sgRNA).

Adding Functionality to Down-Regulate Transcription

Just as organic chemists can design and synthesize small molecules having desired functional properties, molecular biologists can design and produce complex macromolecules having desired functional elements. The latter is nicely exemplified by Gilbert et el., who demonstrated that fusion of dCas9 to transcription factor effector domains having repressive regulatory functions enables efficient transcriptional repression in human (or yeast) cells via sgRNA that target genes of interest.

Taken from Liu et al. (2017)

As depicted below, Gilbert et al. used dCas9 fused to the Krüppel associated box (KRAB) domain, which is a transcriptional repression domain, and Green Fluorescent Protein (GFP) as a reporter gene targeted by sgRNA. They employed RNA-sequencing to quantify the transcriptome of GFP-positive HEK293 cells expressing dCas9-KRAB or a negative control construct. It was shown that CRISPRi is highly specific, as GFP was the only gene that was significantly suppressed by GFP-targeting sgRNA. Averaged data from two independent biological replicates indicated that no gene other than GFP changes by >1.5-fold.

Genome-Scale CRISPRi to Identify Human lncRNA

According to Liu et al., it has not been possible to predict which lncRNA loci are functional or what function they perform. Consequently, there is a need for large-scale, systematic approaches to interrogating the functional contribution of lncRNA loci. This sizeable team of collaborators from various institutions in the San Francisco Bay area, therefore, developed a genome-scale screening platform using CRISPRi with dCas9-KRAB and a library of sgRNA.

Taken from Liu et al. (2017)

As depicted below for the overall approach, they first designed a CRISPRi Non-Coding Library, which targets 16,401 lncRNA genes each with 10 sgRNAs per transcription start site. The required 170,262 sgRNAs were not synthesized chemically, but rather produced intracellularly by first using array-based sgDNA synthesis followed by clonal (i.e. individual sgDNA sequence) incorporation into lentivirus, which in turn were transfected into seven types of cells for screening. More detail on such lentiviral libraries is given in a Footnote at the end of this blog.

As indicated pictorially above, they applied this pooled screening approach to identify lncRNA genes that modify robust cell growth for induced pluripotent stem cells (iPSC) and six well-known, transformed human cell lines (K562, U87, etc.). This led to identification of 499 lncRNA loci that modified cell growth upon CRISPRi targeting.

Interestingly—at least to me—372 (~75%) and 299 (~60%) of these 499 growth-modifying lncRNA loci were distal to a protein coding gene (PCG) or mapped enhancer, respectively. The diagram below, taken from a review by Vance & Ponting, depicts “distal” effects of lncRNA away from PCG between two chromosomes (chr). What “triggers” transcription of the lncRNA from chr A and how it “finds” its cognate PCG on chr B are open and indeed intriguing questions.

Taken from Vance & Ponting (2014)

In addition to these high percentages of distal effects, Liu et al. found the following surprising results with regard to cell-type specificity of lncRNA function:

“Remarkably, 89% of the lncRNA gene hits modified growth in just one of the cell lines tested, and no hits were common to all seven cell lines. Although nearly all of the hit genes were expressed in the cell line in which they exhibited a growth phenotype, expression alone was insufficient to explain the cell type specificity of their function.”

“[Thus,] in contrast to recent studies that found that essential protein-coding genes typically are required across a broad range of cell types, we show that lncRNA function is highly cell type-specific, a finding that has important implications for their involvement in both normal biology and disease.”

Following are some of the major unanswered questions about lncRNA posed in a review I recommend reading for more background on lncRNA:

  • How does the manner in which lncRNAs are transcribed, processed, and regulated differ from that of other RNAs?
  • Are lncRNAs evolutionarily conserved, both in terms of their primary sequences and secondary structures?
  • Are all lncRNAs functional? Which ones have detectable biological functions in cells or in the whole organism?
  • Does the pervasive transcription that generates the lncRNA transcripts play a regulatory role distinct from the steady-state accumulation of the lncRNAs?
  • Can lncRNAs be exploited for clinical applications and therapeutics?

After reading this review, I thought to myself that there are many open questions about lncRNA but no comprehensive answers yet deciphered. When I then checked Google Scholar for items with both “deciphered” and “lncRNA” as terms, I found there were over 1,800 such items. Evidently, there are quite a few authors who, like me, view unknown functions of lncRNA as a cipher. I suspect that much of the now mysterious lncRNA function will eventually be deciphered thanks, in part, to the power of CRISPRi.

Your comments are welcomed.

Footnote

Readers interested in lentiviral sgRNA library construction and use for screening target cells can find general information at this website from which the following self-explanatory schematic provides a high-level overview of the workflow.

Taken from cellecta.com

Lab-on-a-Drone and Other Innovative Point-of-Care Devices

  • Lab-in-a-Box…Think Bento
  • Lab-on-a-Robot…Rolls Along 
  • Lab-on-a-Drone…PCR-on-the-Fly

Honey! I shrunk the lab! 

Taken from gene-quantification.de

Researchers have long dreamed of a “lab-on-chip” (LOC) wherein common laboratory procedures have been miniaturized and integrated in various formats using microfluidics—small, interconnected channels resembling electronic circuits on a chip—that provide low-cost assays for “point-of-care” (POC) applications. The cartoon to the right humorously but concisely depicts the general concept of LOC, for which there are virtually an infinite number of specific embodiments made possible by continuing development of many clever fabrication and microfluidic technologies for “shrinking” lab procedures.

Importantly, lab personnel are thus freed-up from slavish, repetitive tasks to instead carry out discovery and development work. Testament to the significance of LOC is evident from the astounding—to me—130,000 items I found in Google Scholar by searching LOC as an exact-word phrase. There is also a LOC Wikipedia site and a journal for LOC specialists named—appropriately—Lab on a Chip, which is already in its 15th year.

What follows is my take on some of the conceptual morphing, so to speak, of LOC-enabled devices that can be packed for portability, driven by remote control, or flown-“in-and-out” for all manner of unconventional, but critically important POC situations needing nucleic acid-based tests.

Lab-in-a-Box 

In archived blogs I’ve previously commented on examples of commercially available portable POC devices that are variations of a lab-in-a-box that can be easily carried in luggage or a back pack. By way of updates, here are some new applications for these systems illustrating wide diversity of use and location:

  • Ubiquitome’s hand-held qPCR system for molecular testing in New Zealand forests aimed at protecting indigenous Kauri trees—the oldest tree species in the world.
  • Amplyus’ miniPCR system for combating Ebola in villages deep in Sierra Leone, Africa.
  • Oxford Nanopore’s thumb-drive size DNA sequencer to identify organisms in the Canadian high Arctic.

Taken from @WhyteLab

RAZOR system by BioFire Defense. Taken from biofiredefence.com

In the above examples, sample prep workflow is still in need of automation with appropriate LOC technology. However, progress in this regard is being made. One example is the RAZOR system developed by BioFire Defense (pictured below) that features a qPCR lab-in-a-box with ready-to-use, freeze-dried reagent pouches for the detection and identification of pathogens and bio-threat agents. While the progress is impressive, there is still work to be done. A dramatized video for RAZOR usage revealed that much manual manipulation and dexterity with syringes are still required, which suggests the need for complete LOC automation in the future.

Another example of facilitating POC sample prep is the Bento Lab, which is named to be word-play on Bento Box—a complete Japanese lunch in a small, partitioned box-like plate. This portable DNA laboratory created by Bethan Wolfenden and Philipp Boeing at University College London is small enough to fit into a laptop bag, weighs only 6.6 pounds, and can now be preordered for ~$1,000 as a “must have” accessory for so-called “citizen scientists,” some of whom have had early access and have posted their personal Bento Lab stories.

The Bento Lab. Taken from Bento Lab

Lab-on-a-Robot

Biohazard accidents happen, as do bio-threat acts of terrorism. In these seriously scary situations, it may be safer or necessary for first-responders to deploy an Autonomous Vehicle as a self-navigating/driving lab-on-a-robot. Sounds far out, but the first example of a mobile lab-on-a-robot was demonstrated in 2008 by Berg et al., and is pictured below.

Taken from Berg et al. (2008)

This particular lab-on-a-robot is able to autonomously navigate by GPS, acquire an air sample, perform multi-step analysis [i.e. injection, capillary electrophoretic separation, and electrochemical (EC) detection], and send data (electropherogram) to a remote station without exposing an analyst to the testing environment. It’s easy to imagine adapting this kind of robot for carrying out qPCR with EC or fluorescence detection, or nanopore sequencing, for rapidly identifying pathogens.

Lab-on-a-Drone

A logical variation of lab-on-a-robot is to attach the lab part to Unmanned Aerial Systems, (more commonly called drones), thus affording a means for “fly-in, fly-out” applications that require speed to and from a location, or for deployment to otherwise inaccessible locations. This biotech version of drone delivery was initially demonstrated for drone pick-up to aerially transport blood samples from patients to central testing labs, as reported by Amukele et al.

Victor Ugaz. Taken from tamu.edu

The much more difficult task of attaching a lab testing module to a drone has been recently demonstrated by Prof. Victor Ugaz and coworkers at Texas A&M University. Their pioneering 2016 publication titled Lab-on-a-drone: toward pinpoint deployment of smartphone-enabled nucleic acid-based diagnostics for mobile health care is loaded with details, and is a “must read” for technophiles. What follows is my extraction of some unique highlights of that work, as well as information I learned by contacting Prof. Ugaz, who incidentally has received numerous awards and honors.

The basic idea investigated by these researchers was to design a drone-compatible system that could perform what I call “qPCR-on-the-fly.” The drone would require low power consumption and use a smartphone for both fluorescence detection—via its camera—and data analysis via radio transmission of results on-the-fly.

To reduce power consumption by conventional PCR using thermal cycling, which uses power for both heating and cooling during each cycle of amplification, the Texas team invented a radically different approach to achieve isothermal PCR. As depicted below, this new method called convective thermocycling operates isothermally at 95oC and involves movement of reactants upward, away from the heater, through progressively cooler regions and then traveling downward to repeat heating, etc. in a cyclic manner.

Taken from Ugaz & coworkers (2016)

They mimicked POC for an Ebola virus epidemic, which required on-site sample prep and then reverse transcription of viral RNA into cDNA prior to hot start qPCR that is incompatible with convective PCR. The sample prep step was very cleverly achieved using centrifuge adapters that connect to the drone in place of propellers. These centrifuges in turn—pun intended—were fabricated using state-of-the-art 3D printing, and are pictured below.

Taken from Ugaz & coworkers (2016)

The in-flight lab-on-a-drone is pictured below. While in-flight, smartphone-enabled qPCR (as depicted above) takes place during the return trip to home base in order to save time for re-equipping the drone to return to another site, thus increasing overall patient analysis throughput per drone.

Taken from Ugaz & coworkers (2016)

I contacted Prof. Ugaz to ask whether the reverse transcription (RT)-PCR could also be carried out in flight to further automate and increase drone throughput. He replied as follows:

“Many thanks for your interest in our work!  For the purposes of these proof of concept studies, we performed the RT and hot start steps off-device in a conventional thermocycler. However, these steps could straightforwardly be embedded in the portable device.  In principle it should be just a matter of either programming the heater to run through these additional steps (in which case we need to consider the thermal transient between steps, since we are trying to keep the device as simple as possible), or possibly have multiple separate heating zones on the device and have the user physically move the reactor from one to another for each step.  There are multiple possibilities to achieve this that can be explored, and the ‘best’ choice is likely related to the specific application that is envisioned.  But to answer your question, yes this is possible as a relatively straightforward extension of the current design…I have a student who will be working on this during the summer.” 

To my surprise and delight, Prof. Ugaz also informed me of his interest in investigating TriLink’s CleanAmp™ technologies for CleanAmp™ hot start PCR and CleanAmp™ hot start RT-PCR. He said that “[w]e are looking forward to testing this soon and will keep you posted!”

This work by Prof. Ugaz will hopefully lead to encouraging results, and provide a great example of how TriLink CleanAmp™ technologies are enabling both scientific advancement as well as an amazingly interesting, new application such as that in this lab-on-a-drone story.

As always, your comments here are welcomed.

Nanopore Sequencing by Synthesis (Seq-by-Syn)

  • Yet Another Notable Achievement Involving George Church, ‘The Most Interesting Scientist in the World’ 
  • Team of 30 Coauthors Reports Seq-by-Syn with DNA Polymerase-Nanopore Protein Construct on an Integrated Chip
  • Challenging Improvements Needed for Commercial Reality

Prof. George M. Church. Taken from evolutionnews.org

Devotees of my blog will know that I’m prone to word play such as calling myself a “huge” fan of “tiny” nanopores for DNA sequencing, about which I’ve previously opined. They will also recall that I’m an admitted scientific admirer of George Church, who I think is The Most Interesting Scientist in the World.

Having said this, it’s not surprising that I closely follow what’s trending in nanopore sequencing, and also make an attempt to read all of Church’s papers as they get published because they are almost invariably quite interesting, involve “big ideas,” and in some new way are very educational, at least for me. Following are my comments about a recently published paper on nanopore sequencing in venerable Proceedings of the National Academy of Sciences of the United States of America (aka PNAS) wherein Church is the designated corresponding author.

Backstory

The seminal origins and early history of nanopore sequencing have been recently chronicled and criticized—then clarified—in Nature Biotech in several “To the Editor” items, which collectively provide enlightening insights into who did what when, so to speak. Those of us who are ‘Nanoporati’—a clever term tweeted by Nick Lowman—should definitely read those Nature Biotech items. For now, however, I’ll set the stage, as it were, by echoing a bit of what I’ve posted in the past for nanopores.

Patented but prophetic (i.e. no data) methods for nanopore sequencing DNA is actually a relatively old (~20 year) idea posited by Church and other creative visionaries. On the other hand, nanopore sequencing was first reduced to practice commercially not too long ago by Oxford Nanopore Technologies (ONT). Many years of delay between concept and commercialization was due to the need for gradual evolution of lots of “nanopore-ology” and sequencing biochemistry, as well as developing highly sophisticated electronics and complex algorithms for data analysis.

Nanopore Sequencing-by-Scanning (Seq-by-Scan)

Taken from rsc.org

As depicted below, and as can be best seen in a video, ONT’s commercially available MinION Seq-by-Scan system essentially involves threading a strand of DNA through a protein-based nanopore and converting resultant ionic current fluctuations into nucleotide base sequence.

While there are issues with base-calling accuracy, the remarkably small and readily portable MinION provides fast, real-time sequencing results for a wide variety of applications. These included unique or otherwise compelling Point-of-Care analyses, such as pathogen surveillance, which has been achieved in remote geographical locations and even in outer space aboard the International Space Station, as I’ve previously posted.

Nanopore Seq-by-Syn

In contrast to DNA Seq-by-Scan using a nanopore, which is challenged by pore-based differentiation of similarly sized A, G, C, and T bases, DNA Seq-by-Syn has no such limitation as it uses the DNA as a template for base-by-base (i.e. stepwise) detection of enzymatic synthesis of complementary DNA. Various Seq-by-Syn methods and challenges have been discussed elsewhere, and currently available commercial systems include those from Illumina and PacBio. The former employs nucleotides that are reversible terminators equipped with cleavable fluorescent “tags” on each base. The latter detects fluorescently labeled tags on polyphosphates released upon nucleotide incorporation.

The presently featured DNA Seq-by-Syn publication by Stranges et al., which builds upon two earlier reports cited therein, differs from the above approaches by using nanopore-based detection of mass tags rather than fluorescent tags. In principle, mass tags could afford higher accuracy compared to DNA Seq-by-Scan. However, as will now be explained, achieving improved accuracy is far easier said than done.

The general approach taken to demonstrate proof-of-concept for mass-tagged nanopore DNA Seq-by-Syn is depicted below in simplified cartoon form, but involves a true tour de force—in my opinion—of three key technologies. The first is design and synthesis of the nucleotides with appropriate mass tags, which involves very sophisticated chemistry that is best appreciated by reading detailed, extensive supporting information (SI) for Stranges et al. and SI for an earlier publication by Fuller et al. In a nutshell, these nucleotides have 5’-hexaphosphates linked to relatively large mass tags comprised of complex oligonucleotide structures.

Taken from Stranges et al. PNAS 2016

The second area of technical innovation involves attachment of a single molecule of ϕ29 DNA polymerase to each α-hemolysin (αHL) nanopore in such a manner as to retain its enzyme activity and be positioned such that every released mass tag transits through (i.e., is “captured” by) the nanopore leading to base identification by its current signature. As depicted below in two related representations, each of these heteroheptameric pores is comprised of one modified αHL subunit to which a peptidyl SpyTag moiety is attached, and six unmodified αHL subunits. This allows attachment of one ϕ29 DNA molecule modified with a cognate peptidyl SpyCatcher moiety at a predetermined, time-average distance from the pore.

Taken from Stranges et al. PNAS 2016.

The third key area of innovation deals with insertion of the enzyme-pore conjugate into a lipid bilayer residing on a silanized array (aka chip) of 256 Ag/AgCl electrodes such that there is one functional pore per electrode. Interested readers are encouraged to consult the publication for details, as well as check out related fabrication and methods patents that I found by searching Google Scholar.

Representative Results

The first image shown above depicts what base tag-specific detection would ideally look like if each of the four different bases would have a characteristic current-blockage intensity and persistence. In addition, all pores would ideally function similarly. Not surprisingly, given the stochastic nature of single-molecule systems in general, Stranges et al. found less than ideal behavior.

For example, out of 70 single pores obtained, 25 captured two or more tags, whereas only six of those pores showed detectable captures of all four tagged nucleotides. Data obtained for the pore with the most transitions between tag capture levels (i.e. the best results) is shown below, while results for the other five are given in the SI.

Taken from Stranges et al. PNAS 2016

To quote the authors:

“All four characteristic current levels for the tags and transitions between them can be readily distinguished…Homopolymer sequences in the template, and repeated, high-frequency tag capture events of the same nucleotide in the raw sequencing reads were considered a single base for sequence alignment. We recognized 12 clear sequence transitions in a 20-s period. Out of the 12 base transitions observed in the data, 85% match the template strand, showing that this method can produce results that closely align to the template sequence.” 

Interested readers need to consult and carefully read the SI for Stranges et al. regarding the interpretation of the “repeated, high frequency capture events,” such as that exhibited by C in the above current vs. time plot.

All of the above snippets in aggregate suggest to me that, while this huge amount of work has made progress toward one approach to Seq-by-Syn, many improvements will need to be made before achieving a robust system to successfully compete in the commercial sector.

Authorship, Affiliations, and Acknowledgments

The relatively large team of 30 coauthors listed for Stranges et al. include the following numbers of investigators and affiliations: 1 at Arizona State Univ., 4 at Harvard, 11 at Columbia University, and 14 at Genia Technologies, which is a Santa Clara, CA company that was acquired by Roche in 2014, and is part of Roche Sequencing.

Acknowledgments in Stranges et al. refer to support by Genia and NIH Grant R01 HG007415, which I found was awarded to coauthors George M. Church (Harvard), Jingyue Ju (Columbia), and James J. Russo (Columbia). The end of the abstract of this grant reads as follows:

“The nanopore chips will be enhanced and expanded from the current 260 nanopores to over 125,000 using advanced nanofabrication techniques. We will conduct real-time single molecule Nano-SBS on DNA templates with known sequences to test and optimize the overall system. These research and development efforts will lay the foundation for the production of a commercial single molecule electronic DNA sequencing platform, which will enable routine use of sequencing for medical diagnostics and personalized medicine.”

The conflict of interest statement in Stranges et al. indicates that the technology described therein (called “Nanopore SBS”) has been exclusively licensed by Genia, and that specified coauthors are entitled to royalties through this license. In addition, Church is a member of the Scientific Advisory Board of Genia.

Parting Comments

Long gone are the days when government-funded academic researchers thumbed their noses, if you will, at commercial development. Nowadays almost all academics parlay their government grants into university patents that get licensed to companies, usually with some type of corporate involvement of said academics.

I hasten to add that I’m not implying that NIH-funded academic research being a “seed” for corporate profitability is negative—especially in view of its Small Business Innovative Research (SBIR) program—but rather view it as a paradigm shift for the better, as it allows academic creativity to be harnessed into applications that can hopefully greatly benefit society.

In conclusion, and coming back to George Church, who I highlighted in the introduction to this blog, I must say that he might very well be the academic researcher with the longest list of technology transfer, advisory roles, and founded companies—13 to date—according to a public list that is truly mind boggling, at least to me.

As usual, your comments are welcomed.

Postscript

After writing this blog, Roche announced on December 15, 2016 that “it has officially notified Pacific Bioscience (PacBio) of its intention to terminate its [2013] agreement and efforts to develop a sequencing instrument for use in the clinical research and clinical market using their Single Molecule, Real-Time (SMRT®) technology,” about which I have commented previously. The announcement went on to say Roche would instead focus on internal development efforts” and “actively pursue multiple technologies and commercial strategies.” A GenomeWeb headline was more specific:  “Roche Will Focus on Genia’s Nanopore Technology for Dx Market After Ending Deal With PacBio.”

On December 30, 2016 it was reported that the University of California (UC) filed a patent suit against the Chief Technology Officer (CTO) at Genia, and Genia Technologies, claiming the CTO produced key inventions during his time at UC that he later assigned to Genia, but which should have automatically been assigned to UC. Stay tuned…