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.


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

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

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.


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

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.


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…

Top 10 Innovations 2016

  • Sequencing with Sequel, but with a Shocking Surprise
  • Endonuclease CRISPR-Cas9 Makes the Cut Twice—Pun Intended
  • My Predicted 2016 Innovation Winners Made Lots of News

Welcome to my first blog of the New Year, 2017! My New Year’s resolution is to continue to do my best in providing interesting and informative content about what’s trending in nucleic acid research. As in the past, this first blog of the year comments on the Top 10 Innovations in 2016 that were selected by a panel of judges and published last month in The Scientist. Also like the past, you can peruse TriLink’s top products by clicking here.

So, with an imaginary loud flourish of trumpets, read on to learn about the 10 winners, starting with 1st place.

  1. Milo from ProteinSimple enables single-cell Western blotting in a benchtop instrument that allows researchers to search for specific proteins in about 1,000 single cells simultaneously. A titered cell-suspension is pipetted on a 1-by-3-inch glass microscope slide covered by a 30-micron gel-layer dotted with 6,400 microwells. Some wells will remain empty, but ~1,000 will collect individual cells for antibody-based protein analysis, following lysis and other steps, all done automatically. Indeed simple!
  2. ExVive Human Kidney Tissue from Organovo is a replica of the kidney proximal tubule created using 3D bioprinting, which is like uber-trending 3D printing with plastic, but instead uses tiny aggregates of cells. This novel product offers drug developers a reliable means of testing for renal toxicity that is more predictive than traditional cell culture, and avoids animal testing.
  3. Sequel System from Pacific Biosystems is not neither small (see below) nor inexpensive ($350,000), but is nevertheless a third the size and weight—and half the cost—of PacBio’s original long-read, single molecule, real-time (SMRT) sequencer, PacBio RS II, about which I’ve favorably blogged several times in the past. Moreover, Sequel has seven-times the throughput of PacBio RS II.

Taken from

Development of what would become Sequel was announced by PacBio in 2013 as part of a potentially $75M deal with Roche Diagnostics aimed at DNA sequencing-based products for clinical diagnostics. Surprisingly—if not shockingly—in December 2016, after Sequel was launched, Roche stated it was terminating this deal with PacBio in order to focus on its internal development efforts.

At this time, I can only speculate that Roche’s internal efforts include single-molecule DNA sequencing using nanopore technology developed by Genia Technologies, Inc., which Roche acquired in 2014 for $125M—plus even greater contingent payments. By remarkable coincidence, I had been drafting a blog about Genia from a purely technical perspective, but will now update that for posting on January 24th as a sequel to Sequel—pun intended.

  1. Lumos from Axion Biosystems is a 48-well light-delivery device allowing researchers to incorporate cutting-edge optical assay techniques, such as optogenetics, into their in vitro research. Lumos delivers user-specified intensity and duration of light with up to four wavelengths simultaneously for assay flexibility.
  2. LentiArray CRISPR Libraries from Thermo Fisher Scientific make applying lentivirus-encoded CRISPR for gene editing more accessible to researchers. Given the continuing explosive-like interest in DNA endonuclease CRISPR-Cas9, which in 2015 also made the Top 10 cut—pun intended—I was expecting to find this endonuclease system among the 2016 Top 10, too. What surprised me, however, was seeing this system split into two parts—pun intended—i.e. CRISPR per se and separately as Cas9, which you’ll find below.
  3. nCounter Vantage 3D Panels from NanoString utilize an automated microscope that counts color-coded barcodes conjugated to target molecules that are mRNAs, DNAs, proteins, and even phosphorylation status of proteins—all at the same time, which I think is quite a technical achievement. The nCounter analysis system ranges from $149,000 to $280,000, and the nCounter Vantage 3D Panels run from $275 per sample and upward.

Taken from

  1. ZipChip from 908 Devices is a cleverly designed microfluidic chip that radically speeds up sample prep for mass spectrometry, requires only a few microliters of sample volumes, and broadens the range of materials that a mass spectrometer can handle. In less than 3 minutes per sample, ZipChip can process cell growth media, cell lysates, blood, plasma, or urine. The 1-by-3-inch chip is in a small box, less than a foot long, which mounts directly onto a mass spec. The device costs $30,000 and an auto sampler adds another $20,000 to the price.
  2. HAP1 Cells from Horizon have earned a spot in The Scientist Top 10 Innovations for the third year in a row—this time as Turbo GFP Tagged HAP1 Cells, which were selected for their ability to tag proteins of interest with green fluorescent protein (GFP) without requiring that the gene be overexpressed. Turbo GFP cells, which are custom-made using CRISPR-Cas9 gene-editing technology, cost $3,400 and take about 16-18 weeks to develop.

Roger Y. Tsien (February 1, 1952 – August 24, 2016).
Taken from

As a sad side note, Roger Y. Tsien, who was awarded the 2008 Nobel Prize in Chemistry for his discovery and development of GFP, in collaboration with Osamu Shimomura and Martin Chalfie, passed away in 2016 at the age of 64.

  1. Prime sCMOS Camera from Photometrics is a 4.2-megapixel camera that has a built-in algorithm to reduce what is called “shot noise”—the variation inherent in measurements taken using light microscopes—without having to acquire many extra images and then average across them, or increase the light intensity, which can damage samples. The Prime sCMOS camera costs $15,950.
  2. GeneArt Platinum Cas9 Nuclease from Thermo Fisher Scientific is a recombinant Streptococcus pyogenes Cas9 protein purified from E. coli that contains a nuclear localization signal that aids in delivery to target-cell nuclei, where Cas9 works in conjunction with CRISPR. I should add that CRISPR-based gene editing is alternatively achieved by transfection of Cas9 mRNA, which is offered by TriLink, and used as described in a recent exemplary publication by a team of international collaborators.

Revisiting Jerry’s Predictions for 2016 Top 10 Innovations

Devotees of my blog may recall the following predictions I offered in January 2016 as winning innovations-to-be:

  • Direct Genomics in China will resurrect and morph Helicos single-molecule sequencing into a diagnostics instrument.
  • GnuBIO—acquired by Bio-Rad—will offer its long-delayed next-generation sequencing system.

While none of these were selected by The Scientist, they did make news in various ways:

  • Oxford Nanopore’s VolTRAX system for automated sample has very recently been launched. I should also note that its tiny MinION sequencer was rocket-launched—literally—to the International Space Station (ISS) for evaluation in rapid identification of pathogens that might infect astronauts on the ISS, as I’ve commented on in a previous blog. Launch puns intended.
  • Direct Genomics recently announced its GenoCare Analyzer, the world’s first single-molecule genome sequencer that is being engineered exclusively for the clinic, and promises to improve the cost, speed, and quality of clinical genome sequencing by directly reading a patient’s original DNA or RNA molecules without prior amplification.
  • GnuBio now offers a fully integrated sequencing platform which allows users to simply load genomic DNA onto cartridge, place the cartridge into the GnuBIO sequencer, and then press “run.” Within hours, results can be exported directly from the instrument with real-time informatics onboard.

Although my picks weren’t among those in The Scientist list for 2016, I take satisfaction in believing that choosing winning biotechnology products is like art appreciation or judging beauty, both of which are in the eye of the beholder, who can disagree on what their eyes behold.

As usual, your comments are welcomed.

New CRISPR System Reported for Targeting RNA Instead of DNA

  • Current “CRISPR Craze” for DNA Editing is Catalyzing Creativity
  • Early CRISPR Innovator Feng Zhang Now Reports Targeting RNA
  • This New “C2c2” System has Specificity Issues But is Nevertheless Promising

Just when you thought that the “CRISPR craze” would soon transition from the fundamental discovery phase to the improvements phase, something entirely new for CRISPR has come along. That something, recently published by Feng Zhang and others in venerable Science magazine, targets RNA instead of DNA. Consequently, this may lead to transient vs. permanent editing, as well as other RNA- vs. DNA-based applications.

Before further commenting on this exciting new RNA-targeting approach using CRISPR, here are a few snippets about the original DNA version of CRISPR to set the stage, and substantiate my tongue-in-cheek referral to the craze about it.


Taken from

Taken from

Editing with CRISPR, which is short-form for CRISPR-Cas9, uses sequence-specific guide RNA (gRNA) to target DNA for cutting by Cas9 nuclease, as depicted below. Guide RNA and Cas9 can be introduced into cells either encoded in a vector or as synthetic gRNA and synthetic Cas9 mRNA, which TriLink offers in either wild-type or base-modified forms of Cas9 mRNA. CRISPR for genome editing was publicly described in Science in 2012 by co-corresponding authors Jennifer A. Doudna, a biologist at the University of California, Berkeley, and the French microbiologist Emmanuelle Charpentier. But Feng Zhang, at the Broad Institute, was first to obtain a patent on the technique.

Not surprisingly, given the financial potential for DNA editing by CRISPR, which has been called the ‘biotech discovery of the century,’ there is ownership litigation. This dispute is getting rather ugly, if you will, according to an article in Science titled Accusations of errors and deception fly in CRISPR patent fight.

crispPotential financial gain aside, PubMed stats I found clearly substantiate the craze factor in numeric terms: >4,000 publications to date with a rapidly increasing trajectory, i.e. ~600 in 2014 and ~1,200 in 2015, which is an average of roughly 4 publications every day in that year!

By the way, in the chart above that I made for CRISPR publications in PubMed, there was only one report in 2002, which was the first publication to identify CRISPR. These Dutch investigators used computer analysis to find a novel family of repetitive DNA sequences that is present among both domains of the prokaryotes (Archaea and Bacteria), but absent from eukaryotes or viruses. They noted that “[t]his family is characterized by direct repeats, varying in size from 21 to 37 bp, interspaced by similarly sized non-repetitive sequences. To appreciate their characteristic structure, we will refer to this family as the clustered regularly interspaced short palindromic repeats (CRISPR).” 

Taken from

Taken from

CRISPR was also selected as 2015 Science Breakthrough of the Year, and is featured in an interesting YouTube video that is definitely worth watching, in my opinion.

Enough said for CRISPR editing of DNA, let’s move on to RNA editing with CRISPR that offers a fundamentally different editing approach: whereas DNA editing makes permanent changes to the genome of a cell, CRISPR-based RNA-targeting approach may allow researchers to make temporary changes. Moreover, this can be adjusted up or down, and may one day provide greater specificity and functionality than existing methods for RNA interference (RNAi) using either siRNA or antisense oligos.

CRISPR Targeting RNA

Feng Zhang. Taken from

Feng Zhang. Taken from

At the risk of seeming to be too trendy, this section heading could have read “Feng Zhang 2.0” in that Zhang at the Broad Institute, along with co-corresponding author Eugene V. Koonin at NIH and uber-famous “Broadster” Eric Langer plus others on a large team, have characterized a new CRISPR system that targets RNA—but not DNA. In their recent Science publication they demonstrated that this new system involves a Class 2 type VI-A CRISPR-Cas effector—aptly abbreviated C2c2 (pronounced “see too, see too”)—that has RNA-guided RNase function.

The researchers originally identified C2c2 in the bacterium Leptotrichia shahii (L. shahii) in a systematic search for previously unidentified CRISPR systems within diverse bacterial genomes. They focused on C2c2 because its sequence contained two copies of a domain called higher eukaryotes and prokaryotes nucleotide-binding (HEPN) that has only been found in RNases. Mutating the putative catalytic site within either of C2c2’s HEPN domains demonstrated that none of the mutated enzyme versions could cut RNA in vitro, suggesting that both HEPN domains are necessary for C2c2 to work.

CRISPR-C2c2 from L. shahii reconstituted in E. coli to mediate interference of the RNA phage MS2 via crRNA facilitated by the two HEPN nuclease domains. Taken from Abudayyeh et al.

CRISPR-C2c2 from L. shahii reconstituted in E. coli to mediate interference of the RNA phage MS2 via crRNA facilitated by the two HEPN nuclease domains. Taken from Abudayyeh et al.

But C2c2 Cleaves Collateral RNA—a Case for “Lemons into Lemonade”?

Unlike Cas9, which cuts DNA only within the sequence dictated by the CRISPR gRNA, C2c2 was found to make cuts within the target sequence and adjacent, nonspecific sequences. While this collateral cleavage obviously presents a specificity problem, Zhang and his colleagues were able to create a deactivated C2c2 (dC2c2) variant by alanine substitution of any of the four predicted HEPN domain catalytic residues. To me this clever trick is like converting “lemons into lemonade” in that undesired non-specific cleavage is transformed into a programmable RNA-binding protein having potential utility.

For example, the investigators speculate that the ability of dC2c2 to bind to specified sequences could be used in the following ways:

  • Bring effector modules to specific transcripts in order to modulate their function or translation, which could be used for large-scale screening, construction of synthetic regulatory circuits, and other purposes.
  • Fluorescently tag specific RNAs in order to visualize their trafficking and/or localization.
  • Alter RNA localization through domains with affinity for specific subcellular compartments.
  • Capture specific transcripts through direct pull-down of dC2c2 in order to enrich for proximal molecular partners including RNAs and proteins.

Listen to Zhang’s Grad Students 

While the details of this seminal work published in Science is not easily summarized, its practical implications have been concisely translated, if you will, by first coauthor Omar O. Abudayyeh, and second coauthor Jonathan S. Gootenberg—both graduate student members of the Zhang lab—in three short videos that I encourage you to watch at this link.

Left: Omar Abudayyeh. Taken from Right: Jonathan Gootenberg Taken from

Left: Omar Abudayyeh. Taken from Right: Jonathan Gootenberg Taken from

Publication protocol generally lists coauthors in order of contribution, so in this C2c2 publication that has many coauthors, these fellows know what they’re talking about because they did lots of the lab work. Congrats to them, Feng Zhang (again), and all of the other contributors.

As always, your comments are welcomed and encouraged.

RNA World Revisited

  • Scripps Researchers ‘Evolve’ an RNA-Amplifying RNA Polymerase 
  • It’s Used for First Ever All-RNA Amplification Called “riboPCR”
  • TriLink Reagent Plays a Role in this Remarkably Selective in Vitro Evolution Method 
Prof. Gerald Joyce & Dr. David Horning. Photo by Madeline McCurry-Schmidt. Taken from

Prof. Gerald Joyce & Dr. David Horning. Photo by Madeline McCurry-Schmidt. Taken from

Those of you who regularly read my blog will recall an earlier posting on “the RNA World,” which was envisioned by Prof. Walter Gilbert in the 1980s as a prebiotic place billions of years ago when life began without DNA. That post recommended reading more about this intriguing hypothesis by consulting a lengthy review by Prof. Gerald Joyce. Now, Prof. Joyce and postdoc David Horning have advanced the hypothesis one step further by reporting the first ever amplification of RNA by an in vitro-selected RNA polymerase, thus providing significant supportive evidence for the RNA World. Following are their key findings, which were enabled in part by a TriLink reagent—read on to find out which one and how!

In Vitro Evolution of an RNA Polymerase

Horning & Joyce designed an in vitro selection method to chemically “evolve” an RNA polymerase capable of copying a relatively long RNA template with relatively high fidelity. The double emphasis on “relatively” takes into account that the RNA World would have many millions of years to evolve functionally better RNA polymerases capable of copying increasingly longer RNA templates with increasingly higher fidelity.

As depicted below, they started with a synthetic, highly structured ribozyme (black) wherein random mutations were introduced throughout the molecule at a frequency of 10% per nucleotide position to generate a population of 1014 (100,000,000,000,000) distinct variants to initiate the in vitro evolution process. Step 1 involved 5’-5’ click-mediated 1,2,3-trazole (Ø) attachment of an 11-nt RNA primer (magenta) partially complementary to a synthetic 41-nt RNA template (brown) encoding an aptamer that binds guanosine triphosphate (GTP). In Steps 2 and 3, the primer hybridizes to template and is extended by polymerization of A, G, C and U triphosphates (cyan).

Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

GTP aptamer showing red and cyan sequences corresponding to above cartoon. Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

GTP aptamer showing red and cyan sequences corresponding to above cartoon. Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

Step 4 involves binding of aptameric structures to immobilized GTP (green), then photocleavage of the 1,2,3-triazole linkage in Step 5, followed by reverse transcription to cDNA and conventional PCR in Step 6 for transcription into ribozymes in Step 7. Twenty-four rounds of this evolution by selection were carried out, progressively increasing the stringency by increasing the length of RNA to be synthesized by decreasing the time allowed for polymerization. By the 24th round, the population could readily complete the GTP aptamer shown below. Subsequent cloning, sequencing and screening were then used to characterize the most active polymerase, which was designated “24-3.”

The TriLink “Connection”

2'-Azido-dUTP (aka 2'-azido-UTP)

2′-Azido-dUTP (aka 2′-azido-UTP)

The aforementioned in vitro evolution process actually involves tons of experimental details that interested readers will need to consult in the published paper, which is accompanied by an extensive Supporting Information section. In the latter, a subsection titled Primer Extension Reaction describes 3’ biotinylation of the template RNA strand (brown in above scheme) using TriLink “2’-azido-UTP” (more properly named 2’-azido-dUTP) and yeast poly(A) polymerase, followed by click connection of the RNA template’s 3’-terminal 2’-azido moiety to biotin-alkyne. This very clever functionalization of the RNA template strand allowed for subsequent capture of the double-stranded primer extension reaction products on streptavidin-coated beads, followed by elution of the desired nonbiotinylated strand for GTP aptamer selection (Step 4 above).

Properties of RNA Polymerase 24-3

Needless to say—but I will—enzymologists and RNA aficionados will undoubtedly be interested in musing over the kinetic and fidelity properties of RNA polymerase 24-3.

The rate of 24-3 polymerase catalyzed addition to a template-bound primer was measured using an 11-nt template that is cited extensively in the literature to evaluate various ribozymes. It was found that the average rate of primer extension by 24-3 is 1.2 nt/min, which is ∼100-fold faster than that of the starting ribozyme polymerase randomly mutagenized for in vitro selection.

The NTP incorporation fidelities of the starting and 24-3 ribozyme polymerases on this 11-nt test template, at comparable yields of product, are 96.6% and 92.0%, respectively. Horning & Joyce noted that the higher error rate of 24-3 is due primarily to an increased tendency for G•U wobble pairing.

Phenylalanyl tRNA. Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

Phenylalanyl tRNA. Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

Other longer RNA templates having various base compositions or intramolecular structures were also studied, with the stated “final test of polymerase generality” being use of 24-3 to synthesize yeast phenylalanyl tRNA from a 15-nt primer (in red right). The authors humorously describe the results as follows:

“Despite the stable and complex structure of the template, full-length tRNA was obtained in 0.07% yield after 72 h. This RNA product is close to the limit of what can be achieved with the polymerase, but is likely the first time a tRNA molecule has been synthesized by a ribozyme since the end of the RNA world, nearly four billion years ago.”

Exponential Amplification of RNA

PCR is the most widely used method for amplifying nucleic acids, and involves repeated cycles of heat denaturation and primer extension. The 24-3 RNA polymerase was used to carry out PCR-like amplification, but in an all-RNA system (named riboPCR by Horning & Joyce) using A, G, C, and U triphosphates and a 24-nt RNA template composed of two 10-nt primer-binding sites flanking the sequence AGAG. Somewhat special conditions were employed:

  • The concentration of Mg2+ was reduced to minimize spontaneous RNA cleavage
  • PEG8000 was used as a “molecular crowding” agent to improve ribozyme activity at the reduced Mg2+ concentration
  • Tetrapropylammonium chloride was added to lower the melting temperature of the duplex RNA

Under these conditions, 1 nM of the 24-nt RNA template was driven through >40 repeated thermal cycles, resulting in 98 nM newly synthesized template and 106 nM of its complement, corresponding to 100-fold amplification. Sequencing of the amplified products revealed that the central AGAG sequence was largely preserved, albeit with a propensity to mutate the third position from A to G, reflecting the low barrier to wobble pairing.

Amplification of a 20-nt template (without the central insert) was monitored in real time, using FRET from fluorescently labeled primers, and input template concentrations ranging from 10 nM to 1 pM. The resulting amplification profiles shown in the paper are typical for real-time PCR, shifted by a constant number of cycles per log-change in starting template concentration. A plot of cycle-to-threshold vs. logarithm of template concentration, also shown in the paper, was linear across the entire range of dilutions indicating exponential amplification of the template RNA with a per-cycle amplification efficiency of 1.3-fold.

Implications for the Ancient RNA World

It would be an injustice to Horning & Joyce if I would try to paraphrase their concluding discussion of this investigation, so here is what they say:

The vestiges of the late RNA world appear to be shared by all extant life on Earth, most notably in the catalytic center of the ribosome, but most features of RNA-based life likely were lost in the Archaean era. Whatever forms of RNA life existed, they must have had the ability to replicate genetic information and express it as functional molecules. The 24-3 polymerase is the first known ribozyme that is able to amplify RNA and to synthesize complex functional RNAs. To achieve fully autonomous RNA replication, these two activities must be combined and further improved to provide a polymerase ribozyme that can replicate itself and other ribozymes of similar complexity. Such a system could, under appropriate conditions, be capable of self-sustained Darwinian evolution and would constitute a synthetic form of RNA life.

Applications for Today’s World of Biotechnology

The aforementioned report by Horning & Joyce has received wide acclaim in the scientific press and world-wide public media as supporting the existence of a prebiotic RNA World, billions of years ago, from which life on Earth evolved.

While the academic part of my brain, if you will, fully appreciates the significance of these new insights on “living” RNA eons ago, the technical applications part of my brain is more piqued by possible practical uses of all-RNA copying or all-RNA riboPCR.

I, for one, plan to muse over possible applications of such all-RNA systems in today’s world of biotechnology, and hope that you do too, and are willing to share any ideas as comments here.

Virtual Reality for Graphene Nanopores and Space Station Sequencing 

  • Simulated Sequencing Takes Virtual Reality Way Beyond Games  
  • In Silico Simulations Suggest Possible 99.99% Accuracy for Graphene Nanopores 
  • minION Nanopore Sequencer is Sent to the International Space Station



Taken from

This blog is mostly about an international team of researchers who are using Virtual Reality (VR)—in the form of computational modeling—to simulate a new approach to DNA sequencing using nanopores made out of graphene. While VR is a hot trend in all sorts of so-called immersion media, such as those offered by Oculus (that was acquired by Facebook for $2 billion in 2014), computation-based VR has been used by scientists for simulating molecular interactions for a relatively long time. However, extending molecular simulations to complex (aka many-atom) systems like nanopores and DNA has had to wait for bigger, faster, cheaper computing.

In this blog, I’ll also discuss the recent launch of a commercially available nanopore sequencer for the first ever DNA sequencing in space using a self-landing rocket operated by Space X (co-founded by uber-famous multi-billionaire entrepreneur Elon Musk). It’s hard for me to even imagine what seemingly incongruent mix of topics could be more intriguing than these. As the now trendy saying goes, you can’t make this stuff up. But I digress…

Lift off! Taken from                     Self-landing! Taken from indianexpress .com

Lift off! Taken from    –  Self-landing! Taken from

Nanopore Sequencing 

baseFrom an earlier blog you’ll know that I’m a huge fan of tiny nanopores for sequencing, which is a 20+ year old concept, as depicted below from a seminal patent wherein DNA was envisaged as moving through a pore-in-lipid bilayer leading to base-dependent transient blockage of ionic current from which sequence is determined.

Taken from

Taken from

After two decades, this prophetic concept of nanopore sequencing has recently been realized, and commercialized by Oxford Nanopore Technologies (ONT) using “bionanopore” technology. Comparing the images above and below, you’ll see that bionanopores are, in many respects, quite similar to the first described nanopores, wherein a pore-forming protein, α-hemolysin (gray), is embedded in a lipid bilayer (blue). On the other hand, there is an attached DNA-processive enzyme, 29 DNA polymerase (brown), that feeds in the single strand of DNA for sequencing; details may be found elsewhere.

An alternative strategy for nanopore sequencing is to replace this type of bionanopore (composed of biological macromolecules) with a pore constructed of non-biological materials, notably silicon-based semiconductors that enable electrical signal generation and data processing. This would-be evolution of nanopore sequencing from biological constructs to various types of solid-state materials can be read about elsewhere.

Whither Goest Graphene?

It seems the next step in the progression of nanopore technology is those made of graphene—the trivial name for a very special form of carbon that was long known but exceedingly difficult to make. In fact, the process is so difficult that Andre Geim and Kostya Novoselov at The University of Manchester were awarded the 2010 Nobel Prize in Physics for their work enabling the production and characterization of graphene.

Geim and Novoselov. Taken from 

Geim and Novoselov. Taken from

Graphene is a two-dimensional array or “sheet” of carbon atoms that is usually depicted by the ball-and-stick model (pictured at the left below) as a one-atom-thick sheet of otherwise infinite dimensions. Since nothing is infinite in the real world, sheets of graphene have edges to which hydrogen is bonded, but for simplicity is ignored. This carbon-carbon bonding with carbon-hydrogen edges is akin to that in polycyclic aromatic hydrocarbons familiar to readers who are chemists.

Taken from  

Taken from

Taken from Bayley (2010) in

Taken from Bayley (2010) in

Because of graphene’s unique electrical properties and single-atom-thin structure, the basic idea is that a nanometer-size hole in graphene might be made—somehow—to allow DNA and ions to pass through and thus generate electrical signals—somehow—that are accurately deciphered—somehow—into DNA sequence. Oh, and let’s not forget that this sequence information must differentiate—somehow—3’->5’ from 5’->3’ directional pass through. All these “somehows” are meant to indicate that it’s far easier to imagine the concept of graphene nanopore sequencing, as fancifully shown below, than to actually do it.

Taken from Mechant et al. Nano Letters (2010)

Taken from Mechant et al. Nano Letters (2010)

The most daunting practical problem deals with how to “drill” tiny holes in graphene. One approach has been to use controlled electron-beam exposure in a transmission electron microscope. Initial demonstration of this approach was published in 2010 by Merchant et al. in Nano Letters in a paper titled DNA Translocation through Graphene Nanopores, from which the schematic left is taken.

In this device, a few-atoms-layer piece of graphene (1-5 nm thick) having an ~10 nm hole is suspended over a 1 μm diameter hole in a 40 nm thick silicon nitride (SiN) membrane suspended over an ~50 × 50 μm2 aperture in a silicon chip coated with a 5 μm silicon oxide (SiO2) layer in such a way that a bias voltage (VB) is applied between the reservoirs to drive DNA through the nanopore. Although DNA could be detected, the graphene pore size was too big to allow sequence detection.

Taken from Chang et al. Nano Letters (2010)

Taken from Chang et al. Nano Letters (2010)

Similar studies by Schneider et al. were also reported in Nano Letters in 2010, which appears to be a watershed year for this journal inasmuch as another noteworthy nano-detection scheme for DNA was described therein by Chang et al.—but with an important new feature. Namely, using gold electrodes (in yellow, below) separated by only 2 nm and conjugated to dC, a derivative of dG (blue balls) apparently was able to H-bond (magenta) to dC—based on dC-dG complementarity and detected as electron tunneling signals. This transient, base pair-specific H-bonding is what has now been further investigated by others albeit in the following form of Virtual Reality.

Virtual Reality Nanopore Sequencing

In contrast to the above “real” experiments, others have simulated reality using mathematical calculations based on theoretical chemistry, which is Virtual Reality that has physical significance well beyond simply playing games. Mathematical modeling or computation simulations are phrases generally used to describe these so-called in silico “experiments” that serve as indications of what could be done, in theory, if this Virtual Reality is actually translatable to the real world. But I digress…

An international team of investigators in the U.S., Germany, and Netherlands has recently reported studies titled Nucleobase-functionalized graphene nanoribbons for accurate high-speed DNA sequencing. Although this article is a dreaded “pay-to-read” article, there is a brief news piece about it at the website for the U.S. National Institute of Standards and Technology (NIST) where some of this work was conducted.

Taken from

Taken from

As is evident from the schematic shown below, these investigators borrowed from the aforementioned types of publications to imagine a graphene nanopore having its internal edges functionalized with nucleobase moieties that could potentially H-bond with DNA bases in a sequence specific manner—à la Chang et al. Under appropriate conditions, this could provide the basis for sequencing via measurement of induced current fluctuations.

More specifically, they imagined a sheet (aka ribbon) of graphene 4.5 x 5.5 nm with several nucleobase moieties attached to a 2.5 nm nanopore. In animated simulations (which are linked at the NIST website), you can watch how this sensing device would perform at room temperature in water with attached cytosine H-bonding to detect G in DNA.

When you watch this simulation, you’ll immediately notice how “wiggly” DNA is due to random motions of its constituent groups and atoms. You’ll also see detection of each translocating (i.e. passing G) as an increasing signal being recorded in real-time. While time as a parameter in this simulation is real, the simulation itself is not real, but rather virtual reality based on state-of-the art theoretical calculations by a computer.

With that caveat in mind, the performance was said to be 90% accurate (due to missed bases rather than wrongly detecting a base) at a rate of 66 million bases per seconds, which to me is mindboggling ultra-fast. Moreover, if this device could be fabricated as four sequentially-located graphene pores each functionalized with either C, G, A, or T, the researchers estimate that “proofreading” would increase accuracy to 99.99%, as required for sequencing the human genome.

Virtual reality is, well, not reality. And sometimes dreams and reality go in opposite directions. However, if indeed the above imaginary device and simulations were to become reality—nanopore sequencing would indeed be advanced dramatically from today’s performance.

ONT’s minION Sequencer in Space

In transitioning back to reality, it’s almost unbelievable to me that ONT’s minION nanopore sequencer—which I’ve blogged about before—was sent to the International Space Station (ISS) in April 2016 to carry out the first ever sequencing of DNA in space. If that wasn’t enough “buzz”, then the fact that this was achieved by uber-famous Elon Musk’s Space X company made it way more so, along with much ado in successfully landing the rocket’s first-stage on a relatively tiny platform in the ocean. This is all amazing stuff.  And to think that not so long ago, we were thinking how great it would be if self-landing rockets were really possible, not just a fun concept in video games and sci-fi movies!  Maybe virtual reality isn’t that far from becoming reality, but I digress….

The first aim of putting the minION nanopore into space is to demonstrate the feasibility of nanopore sequencing in microgravity. That being done would then allow use of the minION to rapidly sequence astronaut samples in the ISS to diagnose, for example, an infectious disease or other health issue.

Interested readers can peruse elsewhere much more about this historic milestone, as well as watch and listen to a short (but very exciting) video titled Space Station Live: Big DNA Science in a Small Package. The video was posted on Twitter on July 21st and features the minION device (aka The Biomolecular Sequencer).

NASA minION flight hardware for the ISS experiments packaged for shipment to the ISS. Credit: NASA/Sarah Castro. Taken from

NASA minION flight hardware for the ISS experiments packaged for shipment to the ISS. Credit: NASA/Sarah Castro. Taken from

I look forward to learning the results of the minIONs research in space. I hope that it’s “mission accomplished!” and of great use in years to come. BTW, if you’re a “space buff” like me, you can watch and listen to ISS-Mission Control live streaming 24-7 at this website.

As usual, your thoughts about this blog are welcomed as comments.

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.

Death of DNA Dogma?

  • Current Genetic Dogma is DNA → RNA → Protein
  • Two Research Teams Independently Implicate Sperm Short RNA Can Transmit Paternal Genetics
  • More Research Needed to Elaborate the New Dogma

The Central Dogma of all life on Earth is currently understood to be DNA encoding RNA that in turn encodes protein. That genetic inheritance is transferred as DNA was first posited by uber-famous Francis Crick, who coined the term Central Dogma. While dogmatic principles, by definition, should have no exceptions, a few species of viruses can be considered to be exceptional cases in this regard.

The Central Dogma. Taken from

The Central Dogma. Taken from

That said, there is now quite a scientific buzz—if not shudder by some—over reports implicating RNA molecules as direct (i.e. non-DNA) agents for mammalian inheritance. My instantaneous mental responses to these surprising—if not shocking—revelations was first, “Wow, who would have thunk?” and then, “I’ve got to share this news in a blog.” So here it is.

Surprising Science in Sperm

Human sperm. Taken from

Human sperm. Taken from

While most of us are probably at least passingly familiar with textbook descriptions of the basic structure of sperm and its functional role in reproductive molecular biology, more detailed information on its nucleic acid content is less known. Consequently, shown below is a depiction of the basic structural components of a sperm, DNA content, and primary functions for doing its job, so to speak, in fertilization of an egg.

By way of background, here’s information that I thought was worth sharing. My Google Scholar search results for nucleic acid content of sperm included a very impressive technological accomplishment reported by uber-famous professor/entrepreneur Stephen Quake and co-workers in 2012 on microfluidic separation methods for the first ever genome-wide single-cell DNA sequencing of human sperm. Contrary to what one might intuitively expect, 91 genomes of sperm from a single individual were not identical. Since DNA from only one sperm and one egg combine during fertilization, the exact paternal DNA genotypes in the resultant offspring involves “pot luck,” so to speak.

Regarding RNA, my Google Scholar search led to a paper in 2011 by Krawetz et al. on the first ever report of deep-sequencing of short (18-30 bases) RNA (sRNA) in human sperm (for which TriLink offers a high-performance CleanTag™ kit for sRNA library prep as detailed on this poster). Krawetz et al. found microRNA (miRNA) (≈7%), piwi-interacting RNA (piRNA) (≈17%), and repeat-associated sRNA (≈65%). A minor subset of sRNA within the transcription start site/promoter fraction (≈11%) frames the histone promoter-associated regions enriched in genes of early embryonic development. However, reproductive roles for this molecular menagerie (what I tongue-in-cheek call these various sRNAs) remain speculative.

Fast forwarding to present time leads us to the two “wow” publications in venerable Science that triggered this blog:

While you’ll need to read these publications for details, they collectively raise the following controversial question vis-à-vis the Central Dogma for strictly DNA-based inheritance.

Are You Inheriting More Than Genes from Your Father?

Yes, is the surprising—if not bombshell—answer to this question, which I borrowed from Mitch Leslie’s Science editorial headline. If this conclusion is supported by further studies, it forces a fundamental revision of reproductive molecular cell biology. That’s a very big deal, so to speak, with ramifications not to be under appreciated.

Using sRNA library preparation methods analogous to TriLink CleanTag™ for Illumina deep-sequencing, the USA-Canadian team analyzed sperm from male mice fed a low-protein diet, progeny of which showed elevated activity of genes involved in cholesterol and lipid metabolism. They found that >80% of sRNA were fragments from several kinds of transfer RNAs (tRNAs). Most notably, 5′ fragments of tRNA-Gly-CCC, -TCC, and -GCC shown below all exhibited an approximately 2- to 3-fold increase in low-protein sperm.

Arrows indicate ~30- to 34-nt 5′ tRFs. Taken Upasna Sharma et al. Science (2016)

Arrows indicate ~30- to 34-nt 5′ tRFs. Taken Upasna Sharma et al. Science (2016)

To understand when, where, and how these tRNA fragments were formed, as well as unravel functional significance, the researchers describe an experimental tour de force—in my opinion. This included antisense modified-oligonucleotide “knock-out” of these tRNA fragments, as well as “knock-in” injection of <40-nt sRNA populations purified from control and low-protein sperm into control zygotes.

The researchers concluded that the sperm acquired most of these fragments while passing through the epididymis, a duct from the testicle where the cells mature. Functionally, they also link tRNA fragments to regulation of endogenous retro-elements active in the preimplantation embryo.

In the second study, the China-USA team also found tRNA fragments by deep-sequencing of sRNA. After feeding male mice either a high-fat or low-fat diet, the scientists injected the animals’ sperm into unfertilized eggs, and then measured metabolic performance of the offspring, which ate a normal diet. Progeny of fat-eating fathers remained lean; however, they showed two abnormalities often found in their dads and in humans who are obese or diabetic—abnormal absorption of glucose and insensitivity to insulin.

Like the first study, these researchers also did “knock-in” experiments wherein they inserted the tRNA fragments into eggs fertilized with other sperm. Fragments that came from fathers that ate the high-fat diet resulted in offspring that also showed impaired glucose absorption.

Take Home Messages

At the risk of over simplifying or over generalizing, the aforementioned two studies of sRNA in sperm provide compelling—and stunning—evidence for how tRNA fragments in sperm are responsible for inheritance independent of sperm DNA sequences. So much for dogma.

With regard to specifics, researchers now need to investigate how permanent these changes are, and how quickly they can be reversed by changing diet.

The flip-side of a bad diet adversely influencing offspring is to investigate if and how a good diet imparts better health to offspring.

Please share your thoughts about these reports, conclusion, and implications by commenting here.


If you enjoy hip hop music—or just want to chuckle—this YouTube video for the Central Dogma song will get your head bobbing in sync with the music, lead you to smile, and give you a cool visual display of the central dogma.

Nucleic Acid-Based Circulating Biomarkers for Cancer Diagnostics Become Reality

  • Circulating Tumor Cell Blood Tests Approved by FDA
  • Circulating DNA Stool Test Approved for Colorectal Screening to Avoid Colonoscopy
  • Circulating mRNA Urine Test Approved for use to Reduce the Total Number of Unnecessary Prostate Biopsies


Taken from 

Taken from

According to the NIH National Cancer Institute website, ~1.6 million persons in the U.S. alone will be diagnosed with cancer this year. A very important key to survival is early detection. To enable significantly earlier diagnosis compared to manifestation of clinical symptoms, researchers have been focusing on finding DNA or RNA biomarkers that are circulating in blood, which is readily available and relatively noninvasive compared to traditional biopsies.

exosomesSome of the basic processes underlying this paradigm-shift in cancer diagnostics are depicted in the simplified cartoon wherein tumor cells, or components thereof, pass into the bloodstream. This leads to circulating tumor cells (CTCs) and cell-free circulating tumor DNA (ctDNA) to investigate and differentiate from their normal counterparts as sources of potential biomarkers.

That task is much easier said than done because of the need to sort through all of the normal components in blood, as well as deal with circulating cells and DNA derived from apoptosis (aka programed cell death) and necrosis that are normal ongoing “background” to contend with. In addition to CTCs and ctDNA, there is active cellular excretion of small (30-100 nm) exosome particles as depicted in the following graphic. Consequently, gene-encoding mRNAs, gene-regulating micro RNAs (miRNA), and potentially other exosomal components, can serve as diagnostic biomarkers.

Snapshots of Recent Commercial Diagnostic Products

My search of PubMed for publications indexed to “circulating biomarkers” AND “cancer” led to ~9,000 items, the vast majority of which have appeared during the past decade at an accelerating annual rate.  In fact, there were ~1,000 publications in 2014 alone—that’s roughly 3 such publications every day! Those interested in perusing this mountain of information later can use this link, as my intention here is to comment on resultant commercial diagnostic products, each of which provides all-important early diagnosis using a simple blood test, or urine or stool.


In one of my blogs last year, I asserted that liquid biopsies were (metaphorically) clinically valuable “liquid gold” in a modern day Gold Rush. My evidence for the “rush” was a then recent review in Clinical Chemistry stating that “the detection and molecular characterization of CTCs are one of the most active areas of translational cancer research, with >400 clinical studies having included CTCs as a biomarker.” In that vein—double pun intended—who’s struck it rich, so to speak, commercially?

Taken from

Taken from

The answer is Veridex, which developed the CELLSEARCH® CTC Test that has the added distinction of being the first FDA-approved in vitro diagnostic (IVD) test for capturing and counting CTCs to determine the prognosis of patients (in this case for metastatic breast, colorectal or prostate cancer). This test utilizes magnetic capture of cancer-specific antibodies as depicted below.  Veridex was subsequently acquired by Jansen Diagnostics, which now offers a complete system for CELLSEARCH® CTC Test comprising sample collection, sample preparation, and sample analysis using unique immuno-magnetic and fluorescence imaging technology.

In addition, a Swiss molecular diagnostics company, Novigenix, offers its blood tests for early detection of cancer. Colox®, its lead product, is designed to significantly reduce mortality from colorectal cancer through early detection and follow-up colonoscopy. Novigenix’s technology is based on predictive gene expression profiles of circulating blood cells and tumor-derived protein markers.

Taken from Soper and coworkers in Chem. Commun. (2015).

Taken from Soper and coworkers in Chem. Commun. (2015).

Although not yet a diagnostic device, Prof. Steven Soper at UNC-Chapel Hill and a team of coworkers have recently published methods whereby captured CTCs can be enzymatically released for further analysis. This release procedure (depicted right) features use of an oligonucleotide linker containing uracil (U) that is cleaved by USER™, which consists of a mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII.

ctDNA Biomarkers for Colon Cancer Screening

That ctDNA can provide promising biomarkers for noninvasive assessment of cancer has been successfully translated into a commercial product by Trovagene, which tests for ctDNA in urine or blood, and claims to have been the first company to have recognized the diagnostic value of ctDNA.

In addition, Cologuard® (developed by Exact Sciences in Madison, WI) was approved by the FDA as the first stool-based colorectal screening test that detects red blood cells and DNA mutations that may indicate colon cancer or precursors to cancer. Its commercials are frequently seen on TV. Given the inconvenient colon-cleansing required of patients prior to the also unpleasant invasiveness of colonoscopy, it’s not surprising that more and more persons are opting to use this new test.

In fact, Exact Sciences recently reported that during the first quarter of 2016, the company completed approximately 40,000 Cologuard® tests, an increase of more than 260% compared to approximately 11,000 tests completed in the same quarter of 2015. The cumulative number of physicians ordering Cologuard® since launch expanded to more than 32,000. Finding a doctor is relatively easy, as I found out when I located a gastrointestinal (GI) specialist near me who was also in my network—yeh!

Given the high incidence rate of colon cancer, and the traditionally recommended screening process, it was necessary for Exact Sciences to obtain compelling data in a large clinical study. An FDA announcement stated that the safety and effectiveness of Cologuard® was established in a clinical trial that screened 10,023 subjects. The trial compared the performance of Cologuard® to the fecal immunochemical test (FIT), a commonly used non-invasive screening test that detects blood in the stool. Cologuard® accurately detected cancers and advanced adenomas more often than the FIT test.

Other ctDNA Biomarkers

PlasmaSelect-R™ offered by Personal Genomics Diagnostics, which is a service company founded by experts at Johns Hopkins University, analyzes ctDNA in blood for genetic alterations in cancer based on a targeted panel of 63 well-characterized cancer genes. Cell-free DNA is extracted from plasma using proprietary methods for low-abundance sample DNA, and processed using a proprietary capture process for high-coverage next-generation sequencing to allow tumor specific mutations, amplifications, and translocations to be identified with a high sensitivity (allele fractions as low as 0.10%) and specificity. The company states that its “services further the understanding of cancer and facilitate the development of new diagnostics and therapeutics through our pioneering research approaches and novel technologies.” 

In June 2016, Roche announced that the FDA approved the cobas® EGFR Mutation Test v2 for use with plasma samples, as a companion diagnostic for the non-small cell lung cancer (NSCLC) therapy, Tarceva®. It’s important to recognize that this is the first FDA approval of a liquid biopsy test as an aid in clinical decisions, and makes it the only companion diagnostic that is FDA-approved for the detection of the epidermal growth factor receptor (EGFR) gene in tumor DNA derived from plasma (or tumor tissue). NSCLC patients who have EGFR exon 19 deletions or L858R mutations are candidates for the EGFR-targeted therapy Tarceva® (erlotinib) in first-line treatment.

Circulating RNA and miRNA

The discoveries in 1999-2000 of tumor-derived RNA in the blood of cancer patients sparked a new field for studying gene expression noninvasively using quantitative reverse transcription-PCR (qRT-PCR) and then next-generation sequencing. The existence of circulating RNA was surprising because ribonucleases are present in blood. However, mechanisms that protect circulating RNA reportedly include complexation to lipids, proteins, lipoproteins, or nucleosomes, and protection within apoptotic bodies or other vesicular structures.

Cleverly named Molecular Stethoscope is a newish startup co-founded by uber-famous Drs. Stephen Quake and Eric Topol. The company has leveraged Quake’s finding that genome-wide analysis of circulating RNA shows tissue-specific signatures from all of the major organs can be monitored in blood, and Topol’s finding that such signatures can be used to predict imminent occurrence of a heart attack. Coronary artery disease, neurodegenerative diseases, and autoimmune/inflammatory diseases are the company’s current objectives. I’m guessing, however, that cancer might be added or licensed.

My search of the literature indicates that there are far more publications on circulating miRNA, presumably due to its greater abundance resulting from its small size and/or binding to miRNA-related proteins. The biogenesis of miRNA is depicted below.

Taken from

Taken from

A review and prospectus for circulating miRNA applied to cancer has been recently published by Bertoli et al. in an article entitled MicroRNAs: New Biomarkers for Diagnosis, Prognosis, Therapy Prediction and Therapeutic Tools for Breast Cancer. From my search of this emerging field, some exemplary commercial endeavors are as follows.

The first blood-based cancer diagnostic to exploit exosomes became commercially available in the U.S. in January 2016 via launch of ExoDx Prostate(IntelliScore) by Cambridge, MA-based Exosome Diagnostics. As reported by a large team of medical experts in JAMA Oncology, qRT-PCR was used to compare the urine exosome 3-gene expression with biopsy outcomes in patients with a range of low-to-high prostate-specific antigen (PSA) levels (2 to20 ng/mL).

Taken from

Taken from

The investigators concluded that this qRT-PCR assay using urine was associated with improved identification of patients with higher-grade prostate cancer among men with elevated PSA levels and could reduce the total number of unnecessary biopsies from the ~1M total annual biopsies. The complications that have been associated with unnecessary biopsy and overtreatment range from erectile dysfunction and incontinence, to infections, sepsis and serious cardiovascular events.

At the other end of the commercial spectrum, so to speak, startup Miroculus aims to aid in the early diagnosis of cancer by making a low-cost, open-source, decentralized diagnostic they called Miriam pictured below. Their goal is for untrained workers in clinics around the world to be able to use Miriam to screen for cancer.

Taken from

Taken from

Miriam made its—or more gender specific—her public debut at the TEDGlobal conference in Rio De Janeiro in 2014 with TED curator Chris Anderson calling it ‘one of the most thrilling demos in TED history’, according to Miroculus. To see and hear why this opinion is accurate, and how Miriam will work in concert with a smartphone camera and cloud interface, I urge you to check out the ~11 minute TEDGlobal presentation at this link, which also gives a short, layperson introduction to miRNA biomarkers in blood for cancer.

Oh, One More Thing

Taken from

Taken from

Although this post focuses on nucleic acids, it’s worth noting that protein biomarkers in blood are also being investigated. In view of increased awareness and media attention about concussion injuries in the National Football League (NFL), a timely example of protein biomarkers for diagnosis of chronic traumatic encephalopathy (CTE)—which heretofore has not been possible by any test—is in development.

Currently the only way to diagnose CTE is through a post-mortem autopsy, but Aethlon Medical Inc. intends to change that with the diagnostic test being developed by its subsidiary Exosome Sciences. The test being studied is designed to identify an abnormal protein called tau that builds up in brain tissue as a result of repetitive head trauma. CTE researchers believe that they have developed a means of measuring plasma exosomal tau. Researchers thought that exosomes had potential as a means of identifying CTE because they cross the blood-brain barrier and can provide a unique method of measuring certain aspects of the contents of brain cells through a blood test.

Exosome Science was able to use its diagnostic blood test in 78 NFL players with histories of concussions, as well as in a control group made up of 16 athletes involved in non-contact sports. The subjects are all part of a much larger NIH-funded project called DETECT, which is focused on developing a variety of biomarkers for CTE and involves researchers at Boston University School of Medicine and the University of Washington.

Look for a future post here about DETECT involving nucleic acid biomarkers.

As always, your comments are welcomed.

Dietary Intake of Plant miRNA in Humans is Exciting but Controversial

  • Chinese Team Claims Dietary Rice miRNA can Regulate Gene Expression in Humans
  • Collaboration by miRagen Therapeutics and Monsanto Reports Inability to Detect Bioavailability of Dietary Rice miRNA
  • City of Hope Investigators Claim First Evidence for Plant miRNA having Anticancer Activity



Taken from

You’re probably familiar with the adage “you are what you eat,” the origins of which I found attributable to 17th century Europeans. As scientists, we can intuitively understand this concept by thinking about our dietary food as biochemical inputs, and our body content as biochemical outputs. Further intuition suggests that this input/output cuts both ways, so to speak, as stated here by noted healthy food advocate, Ann Wigmore.

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