Tag Archives: CRISPR-Cas9

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 in the forest

It seems lignin is a bit of a problem. Its presence in a tree makes processing the wood into various products more difficult. (Of course, some people appreciate trees for other reasons both practical [carbon sequestration?] and/or aesthetic.)

In any event, scientists have been working on ways to reduce the amount of lignin in poplar trees since at least 2014 (see my April 7, 2014 posting titled ‘Good lignin, bad lignin: Florida researchers use plant waste to create lignin nanotubes while researchers in British Columbia develop trees with less lignin’; scroll down about 40% of the way for the ‘less lignin’ story).

(I don’t believe the 2014 research was accomplished with the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 technique as it had only been developed in 2012.)

The latest in the quest to reduce the amount of lignin of poplar trees comes from a Belgian/US team, from an Oct. 6, 2020 news item on ScienceDaily,

Researchers led by prof. Wout Boerjan (VIB-UGent [Ghent University] Center for Plant Systems Biology) have discovered a way to stably finetune the amount of lignin in poplar by applying CRISPR/Cas9 technology. Lignin is one of the main structural substances in plants and it makes processing wood into, for example, paper difficult. This study is an important breakthrough in the development of wood resources for the production of paper with a lower carbon footprint, biofuels, and other bio-based materials. Their work, in collaboration with VIVES University College (Roeselare, Belgium) and University of Wisconsin (USA) appears in Nature Communications.

Picture Tailoring lignin and growth by creating CCR2 allelic variants (From left to right: wild type, CCR2(-/-), CCR2(-/*) line 206, CCR2(-/*) line 12) Courtesy: VIB (Flanders Institute of Biotechnology)

An Oct. 6, 2020 VIB (Vlaams Instituut voor Biotechnologie; Flanders Institute of Biotechnology) press release (also on EurekAlert), which originated the news item, explains the reason for this research and how CRISPR (clustered regularly interspaced short palindromic repeats) technology could help realize it,

Towards a bio-based economy

Today’s fossil-based economy results in a net increase of CO2 in the Earth’s atmosphere and is a major cause of global climate change. To counter this, a shift towards a circular and bio-based economy is essential. Woody biomass can play a crucial role in such a bio-based economy by serving as a renewable and carbon-neutral resource for the production of many chemicals. Unfortunately, the presence of lignin hinders the processing of wood into bio-based products.

Prof. Wout Boerjan (VIB-UGent): “A few years ago, we performed a field trial with poplars that were engineered to make wood containing less lignin. Most plants showed large improvements in processing efficiency for many possible applications. The downside, however, was that the reduction in lignin accomplished with the technology we used then – RNA interference – was unstable and the trees grew less tall.”

New tools

Undeterred, the researchers went looking for a solution. They employed the recent CRISPR/Cas9 technology in poplar to lower the lignin amount in a stable way, without causing a biomass yield penalty. In other words, the trees grew just as well and as tall as those without genetic changes.

Dr. Barbara De Meester (VIB-UGent): “Poplar is a diploid species, meaning every gene is present in two copies. Using CRISPR/Cas9, we introduced specific changes in both copies of a gene that is crucial for the biosynthesis of lignin. We inactivated one copy of the gene, and only partially inactivated the other. The resulting poplar line had a stable 10% reduction in lignin amount while it grew normally in the greenhouse. Wood from the engineered trees had an up to 41% increase in processing efficiency”.

Dr. Ruben Vanholme (VIB-UGent): “The mutations that we have introduced through CRISPR/Cas9 are similar to those that spontaneously arise in nature. The advantage of the CRISPR/Cas9 method is that the beneficial mutations can be directly introduced into the DNA of highly productive tree varieties in only a fraction of the time it would take by a classical breeding strategy.”

The applications of this method are not only restricted to lignin but might also be useful to engineer other traits in crops, providing a versatile new breeding tool to improve agricultural productivity.

Here’s a link to and a citation for the paper,

Tailoring poplar lignin without yield penalty by combining a null and haploinsufficient CINNAMOYL-CoA REDUCTASE2 allele by Barbara De Meester, Barbara Madariaga Calderón, Lisanne de Vries, Jacob Pollier, Geert Goeminne, Jan Van Doorsselaere, Mingjie Chen, John Ralph, Ruben Vanholme & Wout Boerjan. Nature Communications volume 11, Article number: 5020 (2020) DOI: https://doi.org/10.1038/s41467-020-18822-w Published 06 October 2020

This paper is open access.

Of puke, CRISPR, fruit flies, and monarch butterflies

I’ve never seen an educational institution use a somewhat vulgar slang term such as ‘puke’ before. Especially not in a news release. You’ll find that elsewhere online ‘puke’ has been replaced, in the headline, with the more socially acceptable ‘vomit’.

Since I wanted to catch this historic moment amid concerns that the original version of the news release will disappear, I’m including the entire news release as i saw it on EurekAlert.com (from an October 2, 2019 University of California at Berkeley news release),

News Release 2-Oct-2019

CRISPRed fruit flies mimic monarch butterfly — and could make you puke
Scientists recreate in flies the mutations that let monarch butterfly eat toxic milkweed with impunity

University of California – Berkeley

The fruit flies in Noah Whiteman’s lab may be hazardous to your health.

Whiteman and his University of California, Berkeley, colleagues have turned perfectly palatable fruit flies — palatable, at least, to frogs and birds — into potentially poisonous prey that may cause anything that eats them to puke. In large enough quantities, the flies likely would make a human puke, too, much like the emetic effect of ipecac syrup.

That’s because the team genetically engineered the flies, using CRISPR-Cas9 gene editing, to be able to eat milkweed without dying and to sequester its toxins, just as America’s most beloved butterfly, the monarch, does to deter predators.

This is the first time anyone has recreated in a multicellular organism a set of evolutionary mutations leading to a totally new adaptation to the environment — in this case, a new diet and new way of deterring predators.

Like monarch caterpillars, the CRISPRed fruit fly maggots thrive on milkweed, which contains toxins that kill most other animals, humans included. The maggots store the toxins in their bodies and retain them through metamorphosis, after they turn into adult flies, which means the adult “monarch flies” could also make animals upchuck.

The team achieved this feat by making three CRISPR edits in a single gene: modifications identical to the genetic mutations that allow monarch butterflies to dine on milkweed and sequester its poison. These mutations in the monarch have allowed it to eat common poisonous plants other insects could not and are key to the butterfly’s thriving presence throughout North and Central America.

Flies with the triple genetic mutation proved to be 1,000 times less sensitive to milkweed toxin than the wild fruit fly, Drosophila melanogaster.

Whiteman and his colleagues will describe their experiment in the Oct. 2 [2019] issue of the journal Nature.

Monarch flies

The UC Berkeley researchers created these monarch flies to establish, beyond a shadow of a doubt, which genetic changes in the genome of monarch butterflies were necessary to allow them to eat milkweed with impunity. They found, surprisingly, that only three single-nucleotide substitutions in one gene are sufficient to give fruit flies the same toxin resistance as monarchs.

“All we did was change three sites, and we made these superflies,” said Whiteman, an associate professor of integrative biology. “But to me, the most amazing thing is that we were able to test evolutionary hypotheses in a way that has never been possible outside of cell lines. It would have been difficult to discover this without having the ability to create mutations with CRISPR.”

Whiteman’s team also showed that 20 other insect groups able to eat milkweed and related toxic plants – including moths, beetles, wasps, flies, aphids, a weevil and a true bug, most of which sport the color orange to warn away predators – independently evolved mutations in one, two or three of the same amino acid positions to overcome, to varying degrees, the toxic effects of these plant poisons.

In fact, his team reconstructed the one, two or three mutations that led to each of the four butterfly and moth lineages, each mutation conferring some resistance to the toxin. All three mutations were necessary to make the monarch butterfly the king of milkweed.
Resistance to milkweed toxin comes at a cost, however. Monarch flies are not as quick to recover from upsets, such as being shaken — a test known as “bang” sensitivity.

“This shows there is a cost to mutations, in terms of recovery of the nervous system and probably other things we don’t know about,” Whiteman said. “But the benefit of being able to escape a predator is so high … if it’s death or toxins, toxins will win, even if there is a cost.”

Plant vs. insect

Whiteman is interested in the evolutionary battle between plants and parasites and was intrigued by the evolutionary adaptations that allowed the monarch to beat the milkweed’s toxic defense. He also wanted to know whether other insects that are resistant — though all less resistant than the monarch — use similar tricks to disable the toxin.

“Since plants and animals first invaded land 400 million years ago, this coevolutionary arms race is thought to have given rise to a lot of the plant and animal diversity that we see, because most animals are insects, and most insects are herbivorous: they eat plants,” he said.

Milkweeds and a variety of other plants, including foxglove, the source of digitoxin and digoxin, contain related toxins — called cardiac glycosides — that can kill an elephant and any creature with a beating heart. Foxglove’s effect on the heart is the reason that an extract of the plant, in the genus Digitalis, has been used for centuries to treat heart conditions, and why digoxin and digitoxin are used today to treat congestive heart failure.

These plants’ bitterness alone is enough to deter most animals, but a small minority of insects, including the monarch (Danaus plexippus) and its relative, the queen butterfly (Danaus gilippus), have learned to love milkweed and use it to repel predators.

Whiteman noted that the monarch is a tropical lineage that invaded North America after the last ice age, in part enabled by the three mutations that allowed it to eat a poisonous plant other animals could not, giving it a survival edge and a natural defense against predators.

“The monarch resists the toxin the best of all the insects, and it has the biggest population size of any of them; it’s all over the world,” he said.

The new paper reveals that the mutations had to occur in the right sequence, or else the flies would never have survived the three separate mutational events.

Thwarting the sodium pump

The poisons in these plants, most of them a type of cardenolide, interfere with the sodium/potassium pump (Na+/K+-ATPase) that most of the body’s cells use to move sodium ions out and potassium ions in. The pump creates an ion imbalance that the cell uses to its favor. Nerve cells, for example, transmit signals along their elongated cell bodies, or axons, by opening sodium and potassium gates in a wave that moves down the axon, allowing ions to flow in and out to equilibrate the imbalance. After the wave passes, the sodium pump re-establishes the ionic imbalance.

Digitoxin, from foxglove, and ouabain, the main toxin in milkweed, block the pump and prevent the cell from establishing the sodium/potassium gradient. This throws the ion concentration in the cell out of whack, causing all sorts of problems. In animals with hearts, like birds and humans, heart cells begin to beat so strongly that the heart fails; the result is death by cardiac arrest.

Scientists have known for decades how these toxins interact with the sodium pump: they bind the part of the pump protein that sticks out through the cell membrane, clogging the channel. They’ve even identified two specific amino acid changes or mutations in the protein pump that monarchs and the other insects evolved to prevent the toxin from binding.

But Whiteman and his colleagues weren’t satisfied with this just so explanation: that insects coincidentally developed the same two identical mutations in the sodium pump 14 separate times, end of story. With the advent of CRISPR-Cas9 gene editing in 2012, coinvented by UC Berkeley’s Jennifer Doudna, Whiteman and colleagues Anurag Agrawal of Cornell University and Susanne Dobler of the University of Hamburg in Germany applied to the Templeton Foundation for a grant to recreate these mutations in fruit flies and to see if they could make the flies immune to the toxic effects of cardenolides.

Seven years, many failed attempts and one new grant from the National Institutes of Health later, along with the dedicated CRISPR work of GenetiVision of Houston, Texas, they finally achieved their goal. In the process, they discovered a third critical, compensatory mutation in the sodium pump that had to occur before the last and most potent resistance mutation would stick. Without this compensatory mutation, the maggots died.

Their detective work required inserting single, double and triple mutations into the fruit fly’s own sodium pump gene, in various orders, to assess which ones were necessary. Insects having only one of the two known amino acid changes in the sodium pump gene were best at resisting the plant poisons, but they also had serious side effects — nervous system problems — consistent with the fact that sodium pump mutations in humans are often associated with seizures. However, the third, compensatory mutation somehow reduces the negative effects of the other two mutations.

“One substitution that evolved confers weak resistance, but it is always present and allows for substitutions that are going to confer the most resistance,” said postdoctoral fellow Marianna Karageorgi, a geneticist and evolutionary biologist. “This substitution in the insect unlocks the resistance substitutions, reducing the neurological costs of resistance. Because this trait has evolved so many times, we have also shown that this is not random.”

The fact that one compensatory mutation is required before insects with the most resistant mutation could survive placed a constraint on how insects could evolve toxin resistance, explaining why all 21 lineages converged on the same solution, Whiteman said. In other situations, such as where the protein involved is not so critical to survival, animals might find different solutions.

“This helps answer the question, ‘Why does convergence evolve sometimes, but not other times?'” Whiteman said. “Maybe the constraints vary. That’s a simple answer, but if you think about it, these three mutations turned a Drosophila protein into a monarch one, with respect to cardenolide resistance. That’s kind of remarkable.”

###

The research was funded by the Templeton Foundation and the National Institutes of Health. Co-authors with Whiteman and Agrawal are co-first authors Marianthi Karageorgi of UC Berkeley and Simon Groen, now at New York University; Fidan Sumbul and Felix Rico of Aix-Marseille Université in France; Julianne Pelaez, Kirsten Verster, Jessica Aguilar, Susan Bernstein, Teruyuki Matsunaga and Michael Astourian of UC Berkeley; Amy Hastings of Cornell; and Susanne Dobler of Universität Hamburg in Germany.

Robert Sanders’ Oct. 2, 2019′ news release for the University of California at Berkeley (it’s also been republished as an Oct. 2, 2019 news item on ScienceDaily) has had its headline changed to ‘vomit’ but you’ll find the more vulgar word remains in two locations of the second paragraph of the revised new release.

If you have time, go to the news release on the University of California at Berkeley website just to admire the images that have been embedded in the news release. Here’s one,

Caption: A Drosophila melanogaster “monarch fly” with mutations introduced by CRISPR-Cas9 genome editing (V111, S119 and H122) to the sodium potassium pump, on a wing of a monarch butterfly (Danaus plexippus). Credit & Ccpyright: Julianne Pelaez

Here’s a link to and a citation for the paper,

Genome editing retraces the evolution of toxin resistance in the monarch butterfly by Marianthi Karageorgi, Simon C. Groen, Fidan Sumbul, Julianne N. Pelaez, Kirsten I. Verster, Jessica M. Aguilar, Amy P. Hastings, Susan L. Bernstein, Teruyuki Matsunaga, Michael Astourian, Geno Guerra, Felix Rico, Susanne Dobler, Anurag A. Agrawal & Noah K. Whiteman. Nature (2019) DOI: https://doi.org/10.1038/s41586-019-1610-8 Published 02 October 2019

This paper is behind a paywall.

Words about a word

I’m glad they changed the headline and substituted vomit for puke. I think we need vulgar and/or taboo words to release anger or disgust or other difficult emotions. Incorporating those words into standard language deprives them of that power.

The last word: Genetivision

The company mentioned in the new release, Genetivision, is the place to go for transgenic flies. Here’s a sampling from the their Testimonials webpage,

GenetiVision‘s service has been excellent in the quality and price. The timeliness of its international service has been a big plus. We are very happy with its consistent service and the flies it generates.”
Kwang-Wook Choi, Ph.D.
Department of Biological Sciences
Korea Advanced Institute of Science and Technology


“We couldn’t be happier with GenetiVision. Great prices on both standard P and PhiC31 transgenics, quick turnaround time, and we’re still batting 1000 with transformant success. We used to do our own injections but your service makes it both faster and more cost-effective. Thanks for your service!”
Thomas Neufeld, Ph.D.
Department of Genetics, Cell Biology and Development
University of Minnesota

You can find out more here at the Genetivision website.

Effective safety strategies for CRISPR (clustered regularly interspaced short palindromic repeats) gene drive experiments

It’s very peculiar being able to understand each word individually in clustered regularly interspaced short palindromic repeats (CRISPR) but not being able to puzzle out much meaning other than the widely known ‘it’s a gene editor’.

Regardless, CRISPR is a powerful gene editing tool and that can lead to trouble. Even before CRISPR, we’ve had some genetic accidents. Perhaps the best known is the ‘killer bee’ or Africanized bee (from its Wikepedia entry),

The Africanized bee, also known as the Africanised honey bee, and known colloquially as “killer bee”, is a hybrid of the western honey bee species (Apis mellifera), produced originally by cross-breeding [emphasis mine] of the East African lowland honey bee (A. m. scutellata) with various European honey bees such as the Italian honey bee A. m. ligustica and the Iberian honey bee A. m. iberiensis.

The Africanized honey bee was first introduced to Brazil in 1956 in an effort to increase honey production, but 26 swarms escaped quarantine in 1957 [emphasis mine]. Since then, the hybrid has spread throughout South America and arrived in North America in 1985. Hives were found in South Texas of the United States in 1990.

Africanized bees are typically much more defensive than other varieties of honey bee, and react to disturbances faster than European honey bees. They can chase a person a quarter of a mile (400 m); they have killed some 1,000 humans, with victims receiving ten times more stings than from European honey bees. They have also killed horses and other animals.

Getting back to how powerful CRISPR is, a group of scientists has developed a set of strategies for safeguarding gene drive experiments (from a January 22, 2019 eLife press release also on EurekAlert),

Researchers have demonstrated for the first time how two molecular strategies can safeguard CRISPR gene drive experiments in the lab, according to a study published today in eLife.

Their findings, first reported on bioRxiv, suggest that scientists can effectively use synthetic target sites and split drives to conduct gene drive research, without the worry of causing an accidental spread throughout a natural population.

Gene drives, such as those trialled in malaria mosquitoes, are genetic packages designed to spread among populations. They do this via a process called ‘drive conversion’, where the Cas9 enzyme and a molecule called guide RNA (gRNA) cut at a certain site in the genome. The drive is then copied in when the DNA break is repaired.

“CRISPR-based gene drives have sparked both enthusiasm and deep concerns due to their potential for genetically altering entire species,” explains first author Jackson Champer, Postdoctoral Fellow in the Department of Biological Statistics and Computational Biology at Cornell University, New York. “This raises the question about our ability to prevent the unintended spread of such drives from the laboratory into the natural world.

“Current strategies for avoiding accidental spread involve physically confining drive-containing organisms. However, it is uncertain whether this sufficiently reduces the likelihood of any accidental escape into the wild, given the possibility of human error.”

Two molecular safeguarding strategies have recently been proposed that go beyond simply confining research organisms. The first is synthetic target site drive, which homes into engineered genomic sites that are absent in wild organisms. The second is split drive, where the drive construct lacks a type of enzyme called the endonuclease and relies instead on one engineered into a distant site.

“The nature of these strategies means that they should prevent an efficient spread outside of their respective laboratory lines,” Champer adds. “We wanted to see if they both had a similar performance to standard homing drives, and if they would therefore be suitable substitutes in early gene-drive research.”

To do this, the team designed and tested three synthetic target site drives in the fruit fly Drosophila melanogaster. Each drive targeted an enhanced green fluorescent protein (EGFP) gene introduced at one of three different sites in the genome. For split drives, they designed a drive construct that targeted the X-linked gene yellow and lacked Cas9.

Their analyses revealed that CRISPR gene drives with synthetic target sites such as EGFP show similar behaviour to standard drives, and can therefore be used for most testing in place of these drives. The split drives demonstrated similar performance, and also allow for natural sequences to be targeted in situations where the use of synthetic targets is difficult. These include population-suppression drives that require the targeting of naturally occurring genes

“Based on our findings, we suggest these safeguarding strategies should be adopted consistently in the development and testing of future gene drives,” says senior author Philipp Messer, Assistant Professor in the Department of Biological Statistics and Computational Biology at Cornell University. “This will be important for large-scale cage experiments aimed at improving our understanding of the expected population dynamics of candidate drives. Ultimately, this understanding will be crucial for discussing the feasibility and risks of releasing successful drives into the wild, for example to reduce malaria and other vector-borne diseases.”

Here’s a link to and a citation for the paper,

Molecular safeguarding of CRISPR gene drive experiments by Jackson Champer, Joan Chung, Yoo Lim Lee, Chen Liu, Emily Yang, Zhaoxin Wen, Andrew G Clark, Philipp W Messer. DOI: 10.7554/eLife.41439 Short Report Jan 22, 2019

This paper is open access. For anyone who doesn’t mind reading an earlier version of a paper you can find it at bioRxiv, at https://www.biorxiv.org/content/early/2018/09/08/411876.

elife, which i’ve mentioned here here before in a February 8, 2018 posting is a (from their About eLife webpage)

… non-profit organisation inspired by research funders and led by scientists. Our mission is to help scientists accelerate discovery by operating a platform for research communication that encourages and recognises the most responsible behaviours in science.

Controlling agricultural pests with CRISPR-based technology

CRISPR (clustered regularly interspaced short palindromic repeats) technology is often touted as being ‘precise’, which as far as I can tell, is not exactly the case (see my Nov. 28, 2018 posting about the CRISPR babies [scroll down about 30% of the way for the first hint that CRISPR isn’t]). So, it’s a bit odd to see the word ‘precise’ used as part of a new CRISPR-based technology’s name (from a January 8, 2019 news item on ScienceDaily,

Using the CRISPR gene editing tool, Nikolay Kandul, Omar Akbari and their colleagues at UC San Diego [UC is University of California] and UC Berkeley devised a method of altering key genes that control insect sex determination and fertility.

A description of the new “precision-guided sterile insect technique,” [emphasis mine] or pgSIT, is published Jan. 8 [2019] in the journal Nature Communications.

A January 8, 209 UCSD press release (also on EurekAlert) by Mario Aguilera, which originated the news item, delves further into the research,

When pgSIT-derived eggs are introduced into targeted populations, the researchers report, only adult sterile males emerge, resulting in a novel, environmentally friendly and relatively low-cost method of controlling pest populations in the future.

“CRISPR technology has empowered our team to innovate a new, effective, species-specific, self-limiting, safe and scalable genetic population control technology with remarkable potential to be developed and utilized in a plethora of insect pests and disease vectors,” said Akbari, an assistant professor in UC San Diego’s Division of Biological Sciences. “In the future, we strongly believe this technology will be safely used in the field to suppress and even eradicate target species locally, thereby revolutionizing how insects are managed and controlled going forward.”

Since the 1930s, agricultural researchers have used select methods to release sterile male insects into the wild to control and eradicate pest populations. In the 1950s, a method using irradiated males was implemented in the United States to eliminate the pest species known as the New World Screwworm fly, which consumes animal flesh and causes extensive damage to livestock. Such radiation-based methods were later used in Mexico and parts of Central America and continue today.

Instead of radiation, the new pgSIT (precision-guided sterile insect technique), developed over the past year-and-a-half by Kandul and Akbari in the fruit fly Drosophila, uses CRISPR to simultaneously disrupt key genes that control female viability and male fertility in pest species. pgSIT, the researchers say, results in sterile male progeny with 100 percent efficiency. Because the targeted genes are common to a vast cross-section of insects, the researchers are confident the technology can be applied to a range of insects, including disease-spreading mosquitoes.

The researchers envision a system in which scientists genetically alter and produce eggs of a targeted pest species. The eggs are then shipped to a pest location virtually anywhere in the world, circumventing the need for a production facility on-site. Once the eggs are deployed at the pest location, the researchers say, the newly born sterile males will mate with females in the wild and be incapable of producing offspring, driving down the population.

“This is a novel twist of a very old technology,” said Kandul, an assistant project scientist in UC San Diego’s Division of Biological Sciences. “That novel twist makes it extremely portable from one species to another species to suppress populations of mosquitoes or agricultural pests, for example those that feed on valuable wine grapes.”

The new technology is distinct from continuously self-propagating “gene drive” systems that propagate genetic alterations from generation to generation. Instead, pgSIT is considered a “dead end” since male sterility effectively closes the door on future generations.

“The sterile insect technique is an environmentally safe and proven technology,” [emphasis mine] the researchers note in the paper. “We aimed to develop a novel, safe, controllable, non-invasive genetic CRISPR-based technology that could be transferred across species and implemented worldwide in the short-term to combat wild populations.”

With pgSIT proven in fruit flies, the scientists are hoping to develop the technology in Aedes aegypti, the mosquito species responsible for transmitting dengue fever, Zika, yellow fever and other diseases to millions of people.

“The extension of this work to other insect pests could prove to be a general and very useful strategy to deal with many vector-borne diseases that plague humanity and wreak havoc an agriculture globally,” said Suresh Subramani, global director of the Tata Institute for Genetics and Society.

I have one comment about the ‘safety’ of the sterile insect technique. It’s been safe up until now but, assuming this technique works as described: What happens as this new and more powerful technique is more widely deployed possibly eliminating whole species of insects? Might these ‘pests’ have a heretofore unknown beneficial effect somewhere in the food chain or in an ecosystem? Or, there may be other unintended consequences.

Moving on, here’s a link to and a citation for the paper,

Transforming insect population control with precision guided sterile males with demonstration in flies by Nikolay P. Kandul, Junru Liu, Hector M. Sanchez C., Sean L. Wu, John M. Marshall, & Omar S. Akbari. Nature Communications volume 10, Article number: 84 (2019) DOI: https://doi.org/10.1038/s41467-018-07964-7 Published 08 January 2019

This paper is open access.

The researchers have made this illustrative image available,

Caption: This is a schematic of the new precision-guided sterile insect technique (pgSIT), which uses components of the CRISPR/Cas9 system to disrupt key genes that control female viability and male fertility, resulting in sterile male progeny. Credit: Nikolay Kandul, Akbari Lab, UC San Diego

Lifesaving moths and nanomagnets

Rice University bioengineers use a magnetic field to activate nanoparticle-attached baculoviruses in a tissue. The viruses, which normally infect alfalfa looper moths, are modified to deliver gene-editing DNA code only to cells that are targeted with magnetic field-induced local transduction. Courtesy of the Laboratory of Biomolecular Engineering and Nanomedicine

Kudos to whomever put that diagram together! That’s a lot of well conveyed information.

Now for the details about how this technology might save lives. From a November 13, 2018 news item on Nanowerk,

A new technology that relies on a moth-infecting virus and nanomagnets could be used to edit defective genes that give rise to diseases like sickle cell, muscular dystrophy and cystic fibrosis.

Rice University bioengineer Gang Bao has combined magnetic nanoparticles with a viral container drawn from a particular species of moth to deliver CRISPR/Cas9 payloads that modify genes in a specific tissue or organ with spatial control.

A November 12, 2018 Rice University news release (also on EurekAlert published on November 13, 2018), which originated the news item, provides detail,

Because magnetic fields are simple to manipulate and, unlike light, pass easily through tissue, Bao and his colleagues want to use them to control the expression of viral payloads in target tissues by activating the virus that is otherwise inactivated in blood.

The research appears in Nature Biomedical Engineering. In nature, CRISPR/Cas9 bolsters microbes’ immune systems by recording the DNA of invaders. That gives microbes the ability to recognize and attack returning invaders, but scientists have been racing to adapt CRISPR/Cas9 to repair mutations that cause genetic diseases and to manipulate DNA in laboratory experiments.

CRISPR/Cas9 has the potential to halt hereditary disease – if scientists can get the genome-editing machinery to the right cells inside the body. But roadblocks remain, especially in delivering the gene-editing payloads with high efficiency.

Bao said it will be necessary to edit cells in the body to treat many diseases. “But efficiently delivering genome-editing machinery into target tissue in the body with spatial control remains a major challenge,” Bao said. “Even if you inject the viral vector locally, it can leak to other tissues and organs, and that could be dangerous.”

The delivery vehicle developed by Bao’s group is based on a virus that infects Autographa californica, aka the alfalfa looper, a moth native to North America. The cylindrical baculovirus vector (BV), the payload-carrying part of the virus, is considered large at up to 60 nanometers in diameter and 200-300 nanometers in length. That’s big enough to transport more than 38,000 base pairs of DNA, which is enough to supply multiple gene-editing units to a target cell, Bao said.

He said the inspiration to combine BV and magnetic nanoparticles came from discussions with Rice postdoctoral researcher and co-lead author Haibao Zhu, who learned about the virus during a postdoctoral stint in Singapore but knew nothing about magnetic nanoparticles until he joined the Bao lab. The Rice team had previous experience using iron oxide nanoparticles and an applied magnetic field to open blood vessel walls just enough to let large-molecule drugs pass through.

“We really didn’t know if this would work for gene editing or not, but we thought, ‘worth a shot,'” Bao said.

The researchers use the magnetic nanoparticles to activate BV and deliver gene-editing payloads only where they’re needed. To do this, they take advantage of an immune-system protein called C3 that normally inactivates baculoviruses.

“If we combine BV with magnetic nanoparticles, we can overcome this deactivation by applying the magnetic field,” Bao said. “The beauty is that when we deliver it, gene editing occurs only at the tissue, or the part of the tissue, where we apply the magnetic field.”

Application of the magnetic field allows BV transduction, the payload-delivery process that introduces gene-editing cargo into the target cell. The payload is also DNA, which encodes both a reporter gene and the CRISPR/Cas9 system.

In tests, the BV was loaded with green fluorescent proteins or firefly luciferase. Cells with the protein glowed brightly under a microscope, and experiments showed the magnets were highly effective at targeted delivery of BV cargoes in both cell cultures and lab animals.

Bao noted his and other labs are working on the delivery of CRISPR/Cas9 with adeno-associated viruses (AAV), but he said BV’s capacity for therapeutic cargo is roughly eight times larger. “However, it is necessary to make BV transduction into target cells more efficient,” he said.

Here’s a link to and a citation for the paper,

Spatial control of in vivo CRISPR–Cas9 genome editing via nanomagnets by Haibao Zhu, Linlin Zhang, Sheng Tong, Ciaran M. Lee, Harshavardhan Deshmukh, & Gang Bao. Nature Biomedical Engineering (2018) DOI: https://doi.org/10.1038/s41551-018-0318-7 Published: 12 November 2018

This paper is behind a paywall.

World Science Festival in New York City, May 22 – June 2, 2019

It’s time for the World Science Fair in New York City, which has been around since 2008 according to their About webpage,

The annual live, week-long Festivals, which launched in New York in 2008, have collectively drawn over 2.9 million visitors worldwide, with millions more viewing the programs online. The World Science Festival’s original musical and theatrical works tour nationally and internationally, and March 2016 marked the launch of World Science Festival Brisbane. World Science U is the Foundation’s online education arm where students and lifelong learners can dive more deeply through artfully produced digital education content presented by world-renowned scientists.

I’ve arbitrarily selected three events but there are many more. I notice that several sessions have sold out. From the World Science Festival 2019 events page,

Light Falls: Space, Time, and an Obsession of Einstein

Wednesday, May 22, 2019
7:00 pm – 8:30 pm

Jazz at Lincoln Center’s Frederick P. Rose Hall
May 2019 marks a pivotal milestone in human intellectual history: the 100th anniversary of astronomical observations that confirmed Albert Einstein’s new conception of space, time and gravity–his General Theory of Relativity. In celebration of this momentous achievement, join Brian Greene and an ensemble Broadway cast for Light Falls, an original work for the stage featuring wondrous, fully immersive projections and an original orchestral score, tracing the breakthrough moments, agonizing frustrations, and final emergence into the light as the world’s most intrepid scientific mind took on the universe. And won.
Written by Brian Greene
Music by Jeff Beal
Design by 59 Productions
Directed by Scott Faris
Executive Producer Tracy Day
Sponsored by the Alfred P. Sloan Foundation with additional support from the John Templeton Foundation.
NEW TICKETS JUST RELEASED!
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CRISPR in Context: The New World of Human Genetic Engineering

Tuesday, May 28, 2019
8:00 pm – 9:30 pm

Gerald D. Fischbach Auditorium, Simons Foundation
It’s happened. The first children genetically engineered with the powerful DNA-editing tool called CRISPR-Cas9 have been born to a woman in China. Their altered genes will be passed to their children, and their children’s children. Join CRISPR’s co-discoverer, microbiologist Jennifer Doudna, as we explore the perils and the promise of this powerful technology. It is not the first time human ingenuity has created something capable of doing us great good and great harm. Are we up to the challenge of guiding how CRISPR will shape the future?
Seats are limited and will be made available to registered guests on a first-come, first-served basis. REGISTER NOW!

The Kavli Prize recognizes scientists for their seminal advances in astrophysics, nanoscience, and neuroscience. The series, “The Big, the Small, and the Complex,” is sponsored by The Kavli Foundation.
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….

The Technology that Transforms Us

Thursday, May 30, 2019
7:00 pm – 8:30 pm

NYU Global Center, Grand Hall
We make tools. It defines us. But since the first proto-human tied a stick to a stone, tools have also been making us. Join our panel of philosophers, anthropologists, and futurists as we examine our journey from the stone age to the computer age—seeking clues about who we are, and what we are becoming. Our smartphones have become veritable appendages. How long before we literally merge with our technology? Wearables, implantables, ingestible sensors, digital telepathy, and brain-computer interfaces are all on the horizon. Join us for a fascinating glimpse of a future that is closer than you think.

The Big Ideas Series is supported in part by the John Templeton Foundation.
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This program is sold out. A small number of tickets will be available at the venue 30 minutes prior to the event on a first-come-first-served basis. CLICK HERE to join the waitlist and you’ll be alerted if tickets become available sooner. 
Sold Out

….


The Great Fish Count

Saturday, June 1, 2019
10:00 am – 6:00 pm

Great Fish Count Sites
From Lemon Creek in Staten Island to the shores of the Bronx River, New York’s waterways are teeming with life — and it’s up to you to find it! Led by top marine scientists and biologists in 18 sites across New York’s five boroughs, Westchester, and New Jersey, the Great Fish Count gives attendees of all ages the chance to strap on a pair of waders, cast a net, and discover the underwater world in their own backyard.

This event is FREE and open to the public. RSVP not required, but encouraged. RSVP HERE!

Produced in partnership with the Lamont–Doherty Earth Observatory and the New York State Department of Environmental Conservation

Supported by the Bezos Family Foundation.

Learn More

Free Admission

….

Should you be in New York City during these dates, I hope you’ll get a chance to participate if not the festival or one of its associated events.

Gene editing and personalized medicine: Canada

Back in the fall of 2018 I came across one of those overexcited pieces about personalized medicine and gene editing tha are out there. This one came from an unexpected source, an author who is a “PhD Scientist in Medical Science (Blood and Vasculature” (from Rick Gierczak’s LinkedIn profile).

It starts our promisingly enough although I’m beginning to dread the use of the word ‘precise’  where medicine is concerned, (from a September 17, 2018 posting on the Science Borealis blog by Rick Gierczak (Note: Links have been removed),

CRISPR-Cas9 technology was accidentally discovered in the 1980s when scientists were researching how bacteria defend themselves against viral infection. While studying bacterial DNA called clustered regularly interspaced short palindromic repeats (CRISPR), they identified additional CRISPR-associated (Cas) protein molecules. Together, CRISPR and one of those protein molecules, termed Cas9, can locate and cut precise regions of bacterial DNA. By 2012, researchers understood that the technology could be modified and used more generally to edit the DNA of any plant or animal. In 2015, the American Association for the Advancement of Science chose CRISPR-Cas9 as science’s “Breakthrough of the Year”.

Today, CRISPR-Cas9 is a powerful and precise gene-editing tool [emphasis mine] made of two molecules: a protein that cuts DNA (Cas9) and a custom-made length of RNA that works like a GPS for locating the exact spot that needs to be edited (CRISPR). Once inside the target cell nucleus, these two molecules begin editing the DNA. After the desired changes are made, they use a repair mechanism to stitch the new DNA into place. Cas9 never changes, but the CRISPR molecule must be tailored for each new target — a relatively easy process in the lab. However, it’s not perfect, and occasionally the wrong DNA is altered [emphasis mine].

Note that Gierczak makes a point of mentioning that CRISPR/Cas9 is “not perfect.” And then, he gets excited (Note: Links have been removed),

CRISPR-Cas9 has the potential to treat serious human diseases, many of which are caused by a single “letter” mutation in the genetic code (A, C, T, or G) that could be corrected by precise editing. [emphasis mine] Some companies are taking notice of the technology. A case in point is CRISPR Therapeutics, which recently developed a treatment for sickle cell disease, a blood disorder that causes a decrease in oxygen transport in the body. The therapy targets a special gene called fetal hemoglobin that’s switched off a few months after birth. Treatment involves removing stem cells from the patient’s bone marrow and editing the gene to turn it back on using CRISPR-Cas9. These new stem cells are returned to the patient ready to produce normal red blood cells. In this case, the risk of error is eliminated because the new cells are screened for the correct edit before use.

The breakthroughs shown by companies like CRISPR Therapeutics are evidence that personalized medicine has arrived. [emphasis mine] However, these discoveries will require government regulatory approval from the countries where the treatment is going to be used. In the US, the Food and Drug Administration (FDA) has developed new regulations allowing somatic (i.e., non-germ) cell editing and clinical trials to proceed. [emphasis mine]

The potential treatment for sickle cell disease is exciting but Gierczak offers no evidence that this treatment or any unnamed others constitute proof that “personalized medicine has arrived.” In fact, Goldman Sachs, a US-based investment bank, makes the case that it never will .

Cost/benefit analysis

Edward Abrahams, president of the Personalized Medicine Coalition (US-based), advocates for personalized medicine while noting in passing, market forces as represented by Goldman Sachs in his May 23, 2018 piece for statnews.com (Note: A link has been removed),

One of every four new drugs approved by the Food and Drug Administration over the last four years was designed to become a personalized (or “targeted”) therapy that zeros in on the subset of patients likely to respond positively to it. That’s a sea change from the way drugs were developed and marketed 10 years ago.

Some of these new treatments have extraordinarily high list prices. But focusing solely on the cost of these therapies rather than on the value they provide threatens the future of personalized medicine.

… most policymakers are not asking the right questions about the benefits of these treatments for patients and society. Influenced by cost concerns, they assume that prices for personalized tests and treatments cannot be justified even if they make the health system more efficient and effective by delivering superior, longer-lasting clinical outcomes and increasing the percentage of patients who benefit from prescribed treatments.

Goldman Sachs, for example, issued a report titled “The Genome Revolution.” It argues that while “genome medicine” offers “tremendous value for patients and society,” curing patients may not be “a sustainable business model.” [emphasis mine] The analysis underlines that the health system is not set up to reap the benefits of new scientific discoveries and technologies. Just as we are on the precipice of an era in which gene therapies, gene-editing, and immunotherapies promise to address the root causes of disease, Goldman Sachs says that these therapies have a “very different outlook with regard to recurring revenue versus chronic therapies.”

Let’s just chew on this one (contemplate)  for a minute”curing patients may not be ‘sustainable business model’!”

Coming down to earth: policy

While I find Gierczak to be over-enthused, he, like Abrahams, emphasizes the importance of new policy, in his case, the focus is Canadian policy. From Gierczak’s September 17, 2018 posting (Note: Links have been removed),

In Canada, companies need approval from Health Canada. But a 2004 law called the Assisted Human Reproduction Act (AHR Act) states that it’s a criminal offence “to alter the genome of a human cell, or in vitroembryo, that is capable of being transmitted to descendants”. The Actis so broadly written that Canadian scientists are prohibited from using the CRISPR-Cas9 technology on even somatic cells. Today, Canada is one of the few countries in the world where treating a disease with CRISPR-Cas9 is a crime.

On the other hand, some countries provide little regulatory oversight for editing either germ or somatic cells. In China, a company often only needs to satisfy the requirements of the local hospital where the treatment is being performed. And, if germ-cell editing goes wrong, there is little recourse for the future generations affected.

The AHR Act was introduced to regulate the use of reproductive technologies like in vitrofertilization and research related to cloning human embryos during the 1980s and 1990s. Today, we live in a time when medical science, and its role in Canadian society, is rapidly changing. CRISPR-Cas9 is a powerful tool, and there are aspects of the technology that aren’t well understood and could potentially put patients at risk if we move ahead too quickly. But the potential benefits are significant. Updated legislation that acknowledges both the risks and current realities of genomic engineering [emphasis mine] would relieve the current obstacles and support a path toward the introduction of safe new therapies.

Criminal ban on human gene-editing of inheritable cells (in Canada)

I had no idea there was a criminal ban on the practice until reading this January 2017 editorial by Bartha Maria Knoppers, Rosario Isasi, Timothy Caulfield, Erika Kleiderman, Patrick Bedford, Judy Illes, Ubaka Ogbogu, Vardit Ravitsky, & Michael Rudnicki for (Nature) npj Regenerative Medicine (Note: Links have been removed),

Driven by the rapid evolution of gene editing technologies, international policy is examining which regulatory models can address the ensuing scientific, socio-ethical and legal challenges for regenerative and personalised medicine.1 Emerging gene editing technologies, including the CRISPR/Cas9 2015 scientific breakthrough,2 are powerful, relatively inexpensive, accurate, and broadly accessible research tools.3 Moreover, they are being utilised throughout the world in a wide range of research initiatives with a clear eye on potential clinical applications. Considering the implications of human gene editing for selection, modification and enhancement, it is time to re-examine policy in Canada relevant to these important advances in the history of medicine and science, and the legislative and regulatory frameworks that govern them. Given the potential human reproductive applications of these technologies, careful consideration of these possibilities, as well as ethical and regulatory scrutiny must be a priority.4

With the advent of human embryonic stem cell research in 1978, the birth of Dolly (the cloned sheep) in 1996 and the Raelian cloning hoax in 2003, the environment surrounding the enactment of Canada’s 2004 Assisted Human Reproduction Act (AHRA) was the result of a decade of polarised debate,5 fuelled by dystopian and utopian visions for future applications. Rightly or not, this led to the AHRA prohibition on a wide range of activities, including the creation of embryos (s. 5(1)(b)) or chimeras (s. 5(1)(i)) for research and in vitro and in vivo germ line alterations (s. 5(1)(f)). Sanctions range from a fine (up to $500,000) to imprisonment (up to 10 years) (s. 60 AHRA).

In Canada, the criminal ban on gene editing appears clear, the Act states that “No person shall knowingly […] alter the genome of a cell of a human being or in vitro embryo such that the alteration is capable of being transmitted to descendants;” [emphases mine] (s. 5(1)(f) AHRA). This approach is not shared worldwide as other countries such as the United Kingdom, take a more regulatory approach to gene editing research.1 Indeed, as noted by the Law Reform Commission of Canada in 1982, criminal law should be ‘an instrument of last resort’ used solely for “conduct which is culpable, seriously harmful, and generally conceived of as deserving of punishment”.6 A criminal ban is a suboptimal policy tool for science as it is inflexible, stifles public debate, and hinders responsiveness to the evolving nature of science and societal attitudes.7 In contrast, a moratorium such as the self-imposed research moratorium on human germ line editing called for by scientists in December 20158 can at least allow for a time limited pause. But like bans, they may offer the illusion of finality and safety while halting research required to move forward and validate innovation.

On October 1st, 2016, Health Canada issued a Notice of Intent to develop regulations under the AHRA but this effort is limited to safety and payment issues (i.e. gamete donation). Today, there is a need for Canada to revisit the laws and policies that address the ethical, legal and social implications of human gene editing. The goal of such a critical move in Canada’s scientific and legal history would be a discussion of the right of Canadians to benefit from the advancement of science and its applications as promulgated in article 27 of the Universal Declaration of Human Rights9 and article 15(b) of the International Covenant on Economic, Social and Cultural Rights,10 which Canada has signed and ratified. Such an approach would further ensure the freedom of scientific endeavour both as a principle of a liberal democracy and as a social good, while allowing Canada to be engaged with the international scientific community.

Even though it’s a bit old, I still recommend reading the open access editorial in full, if you have the time.

One last thing abut the paper, the acknowledgements,

Sponsored by Canada’s Stem Cell Network, the Centre of Genomics and Policy of McGill University convened a ‘think tank’ on the future of human gene editing in Canada with legal and ethics experts as well as representatives and observers from government in Ottawa (August 31, 2016). The experts were Patrick Bedford, Janetta Bijl, Timothy Caulfield, Judy Illes, Rosario Isasi, Jonathan Kimmelman, Erika Kleiderman, Bartha Maria Knoppers, Eric Meslin, Cate Murray, Ubaka Ogbogu, Vardit Ravitsky, Michael Rudnicki, Stephen Strauss, Philip Welford, and Susan Zimmerman. The observers were Geneviève Dubois-Flynn, Danika Goosney, Peter Monette, Kyle Norrie, and Anthony Ridgway.

Competing interests

The authors declare no competing interests.

Both McGill and the Stem Cell Network pop up again. A November 8, 2017 article about the need for new Canadian gene-editing policies by Tom Blackwell for the National Post features some familiar names (Did someone have a budget for public relations and promotion?),

It’s one of the most exciting, and controversial, areas of health science today: new technology that can alter the genetic content of cells, potentially preventing inherited disease — or creating genetically enhanced humans.

But Canada is among the few countries in the world where working with the CRISPR gene-editing system on cells whose DNA can be passed down to future generations is a criminal offence, with penalties of up to 10 years in jail.

This week, one major science group announced it wants that changed, calling on the federal government to lift the prohibition and allow researchers to alter the genome of inheritable “germ” cells and embryos.

The potential of the technology is huge and the theoretical risks like eugenics or cloning are overplayed, argued a panel of the Stem Cell Network.

The step would be a “game-changer,” said Bartha Knoppers, a health-policy expert at McGill University, in a presentation to the annual Till & McCulloch Meetings of stem-cell and regenerative-medicine researchers [These meetings were originally known as the Stem Cell Network’s Annual General Meeting {AGM}]. [emphases mine]

“I’m completely against any modification of the human genome,” said the unidentified meeting attendee. “If you open this door, you won’t ever be able to close it again.”

If the ban is kept in place, however, Canadian scientists will fall further behind colleagues in other countries, say the experts behind the statement say; they argue possible abuses can be prevented with good ethical oversight.

“It’s a human-reproduction law, it was never meant to ban and slow down and restrict research,” said Vardit Ravitsky, a University of Montreal bioethicist who was part of the panel. “It’s a sort of historical accident … and now our hands are tied.”

There are fears, as well, that CRISPR could be used to create improved humans who are genetically programmed to have certain facial or other features, or that the editing could have harmful side effects. Regardless, none of it is happening in Canada, good or bad.

In fact, the Stem Cell Network panel is arguably skirting around the most contentious applications of the technology. It says it is asking the government merely to legalize research for its own sake on embryos and germ cells — those in eggs and sperm — not genetic editing of embryos used to actually get women pregnant.

The highlighted portions in the last two paragraphs of the excerpt were written one year prior to the claims by a Chinese scientist that he had run a clinical trial resulting in gene-edited twins, Lulu and Nana. (See my my November 28, 2018 posting for a comprehensive overview of the original furor). I have yet to publish a followup posting featuring the news that the CRISPR twins may have been ‘improved’ more extensively than originally realized. The initial reports about the twins focused on an illness-related reason (making them HIV ‘immune’) but made no mention of enhanced cognitive skills a side effect of eliminating the gene that would make them HIV ‘immune’. To date, the researcher has not made the bulk of his data available for an in-depth analysis to support his claim that he successfully gene-edited the twins. As well, there were apparently seven other pregnancies coming to term as part of the researcher’s clinical trial and there has been no news about those births.

Risk analysis innovation

Before moving onto the innovation of risk analysis, I want to focus a little more on at least one of the risks that gene-editing might present. Gierczak noted that CRISPR/Cas9 is “not perfect,” which acknowledges the truth but doesn’t convey all that much information.

While the terms ‘precision’ and ‘scissors’ are used frequently when describing the CRISPR technique, scientists actually mean that the technique is significantly ‘more precise’ than other techniques but they are not referencing an engineering level of precision. As for the ‘scissors’, it’s an analogy scientists like to use but in fact CRISPR is not as efficient and precise as a pair of scissors.

Michael Le Page in a July 16, 2018 article for New Scientist lays out some of the issues (Note: A link has been removed),

A study of CRIPSR suggests we shouldn’t rush into trying out CRISPR genome editing inside people’s bodies just yet. The technique can cause big deletions or rearrangements of DNA [emphasis mine], says Allan Bradley of the Wellcome Sanger Institute in the UK, meaning some therapies based on CRISPR may not be quite as safe as we thought.

The CRISPR genome editing technique is revolutionising biology, enabling us to create new varieties of plants and animals and develop treatments for a wide range of diseases.

The CRISPR Cas9 protein works by cutting the DNA of a cell in a specific place. When the cell repairs the damage, a few DNA letters get changed at this spot – an effect that can be exploited to disable genes.

At least, that’s how it is supposed to work. But in studies of mice and human cells, Bradley’s team has found that in around a fifth of cells, CRISPR causes deletions or rearrangements more than 100 DNA letters long. These surprising changes are sometimes thousands of letters long.

“I do believe the findings are robust,” says Gaetan Burgio of the Australian National University, an expert on CRISPR who has debunked previous studies questioning the method’s safety. “This is a well-performed study and fairly significant.”

I covered the Bradley paper and the concerns in a July 17, 2018 posting ‘The CRISPR ((clustered regularly interspaced short palindromic repeats)-CAS9 gene-editing technique may cause new genetic damage kerfuffle‘. (The ‘kerfufle’ was in reference to a report that the CRISPR market was affected by the publication of Bradley’s paper.)

Despite Health Canada not moving swiftly enough for some researchers, they have nonetheless managed to release an ‘outcome’ report about a consultation/analysis started in October 2016. Before getting to the consultation’s outcome, it’s interesting to look at how the consultation’s call for response was described (from Health Canada’s Toward a strengthened Assisted Human Reproduction Act ; A Consultation with Canadians on Key Policy Proposals webpage),

In October 2016, recognizing the need to strengthen the regulatory framework governing assisted human reproduction in Canada, Health Canada announced its intention to bring into force the dormant sections of the Assisted Human Reproduction Act  and to develop the necessary supporting regulations.

This consultation document provides an overview of the key policy proposals that will help inform the development of regulations to support bringing into force Section 10, Section 12 and Sections 45-58 of the Act. Specifically, the policy proposals describe the Department’s position on the following:

Section 10: Safety of Donor Sperm and Ova

  • Scope and application
  • Regulated parties and their regulatory obligations
  • Processing requirements, including donor suitability assessment
  • Record-keeping and traceability

Section 12: Reimbursement

  • Expenditures that may be reimbursed
  • Process for reimbursement
  • Creation and maintenance of records

Sections 45-58: Administration and Enforcement

  • Scope of the administration and enforcement framework
  • Role of inspectors designated under the Act

The purpose of the document is to provide Canadians with an opportunity to review the policy proposals and to provide feedback [emphasis mine] prior to the Department finalizing policy decisions and developing the regulations. In addition to requesting stakeholders’ general feedback on the policy proposals, the Department is also seeking input on specific questions, which are included throughout the document.

It took me a while to find the relevant section (in particular, take note of ‘Federal Regulatory Oversight’),

3.2. AHR in Canada Today

Today, an increasing number of Canadians are turning to AHR technologies to grow or build their families. A 2012 Canadian studyFootnote 1 found that infertility is on the rise in Canada, with roughly 16% of heterosexual couples experiencing infertility. In addition to rising infertility, the trend of delaying marriage and parenthood, scientific advances in cryopreserving ova, and the increasing use of AHR by LGBTQ2 couples and single parents to build a family are all contributing to an increase in the use of AHR technologies.

The growing use of reproductive technologies by Canadians to help build their families underscores the need to strengthen the AHR Act. While the approach to regulating AHR varies from country to country, Health Canada has considered international best practices and the need for regulatory alignment when developing the proposed policies set out in this document. …

3.2.1 Federal Regulatory Oversight

Although the scope of the AHR Act was significantly reduced in 2012 and some of the remaining sections have not yet been brought into force, there are many important sections of the Act that are currently administered and enforced by Health Canada, as summarized generally below:

Section 5: Prohibited Scientific and Research Procedures
Section 5 prohibits certain types of scientific research and clinical procedures that are deemed unacceptable, including: human cloning, the creation of an embryo for non-reproductive purposes, maintaining an embryo outside the human body beyond the fourteenth day, sex selection for non-medical reasons, altering the genome in a way that could be transmitted to descendants, and creating a chimera or a hybrid. [emphasis mine]

….

It almost seems as if the they were hiding the section that broached the human gene-editing question. It doesn’t seem to have worked as it appears, there are some very motivated parties determined to reframe the discussion. Health Canada’s ‘outocme’ report, published March 2019, What we heard: A summary of scanning and consultations on what’s next for health product regulation reflects the success of those efforts,

1.0 Introduction and Context

Scientific and technological advances are accelerating the pace of innovation. These advances are increasingly leading to the development of health products that are better able to predict, define, treat, and even cure human diseases. Globally, many factors are driving regulators to think about how to enable health innovation. To this end, Health Canada has been expanding beyond existing partnerships and engaging both domestically and internationally. This expanding landscape of products and services comes with a range of new challenges and opportunities.

In keeping up to date with emerging technologies and working collaboratively through strategic partnerships, Health Canada seeks to position itself as a regulator at the forefront of health innovation. Following the targeted sectoral review of the Health and Biosciences Sector Regulatory Review consultation by the Treasury Board Secretariat, Health Canada held a number of targeted meetings with a broad range of stakeholders.

This report outlines the methodologies used to look ahead at the emerging health technology environment, [emphasis mine] the potential areas of focus that resulted, and the key findings from consultations.

… the Department identified the following key drivers that are expected to shape the future of health innovation:

  1. The use of “big data” to inform decision-making: Health systems are generating more data, and becoming reliant on this data. The increasing accuracy, types, and volume of data available in real time enable automation and machine learning that can forecast activity, behaviour, or trends to support decision-making.
  2. Greater demand for citizen agency: Canadians increasingly want and have access to more information, resources, options, and platforms to manage their own health (e.g., mobile apps, direct-to-consumer services, decentralization of care).
  3. Increased precision and personalization in health care delivery: Diagnostic tools and therapies are increasingly able to target individual patients with customized therapies (e.g., individual gene therapy).
  4. Increased product complexity: Increasingly complex products do not fit well within conventional product classifications and standards (e.g., 3D printing).
  5. Evolving methods for production and distribution: In some cases, manufacturers and supply chains are becoming more distributed, challenging the current framework governing production and distribution of health products.
  6. The ways in which evidence is collected and used are changing: The processes around new drug innovation, research and development, and designing clinical trials are evolving in ways that are more flexible and adaptive.

With these key drivers in mind, the Department selected the following six emerging technologies for further investigation to better understand how the health product space is evolving:

  1. Artificial intelligence, including activities such as machine learning, neural networks, natural language processing, and robotics.
  2. Advanced cell therapies, such as individualized cell therapies tailor-made to address specific patient needs.
  3. Big data, from sources such as sensors, genetic information, and social media that are increasingly used to inform patient and health care practitioner decisions.
  4. 3D printing of health products (e.g., implants, prosthetics, cells, tissues).
  5. New ways of delivering drugs that bring together different product lines and methods (e.g., nano-carriers, implantable devices).
  6. Gene editing, including individualized gene therapies that can assist in preventing and treating certain diseases.

Next, to test the drivers identified and further investigate emerging technologies, the Department consulted key organizations and thought leaders across the country with expertise in health innovation. To this end, Health Canada held seven workshops with over 140 representatives from industry associations, small-to-medium sized enterprises and start-ups, larger multinational companies, investors, researchers, and clinicians in Ottawa, Toronto, Montreal, and Vancouver. [emphases mine]

The ‘outocme’ report, ‘What we heard …’, is well worth reading in its entirety; it’s about 9 pp.

I have one comment, ‘stakeholders’ don’t seem to include anyone who isn’t “from industry associations, small-to-medium sized enterprises and start-ups, larger multinational companies, investors, researchers, and clinician” or from “Ottawa, Toronto, Montreal, and Vancouver.” Aren’t the rest of us stakeholders?

Innovating risk analysis

This line in the report caught my eye (from Health Canada’s Toward a strengthened Assisted Human Reproduction Act ; A Consultation with Canadians on Key Policy Proposals webpage),

There is increasing need to enable innovation in a flexible, risk-based way, with appropriate oversight to ensure safety, quality, and efficacy. [emphases mine]

It reminded me of the 2019 federal budget (from my March 22, 2019 posting). One comment before proceeding, regulation and risk are tightly linked and, so, by innovating regulation they are by exttension alos innovating risk analysis,

… Budget 2019 introduces the first three “Regulatory Roadmaps” to specifically address stakeholder issues and irritants in these sectors, informed by over 140 responses [emphasis mine] from businesses and Canadians across the country, as well as recommendations from the Economic Strategy Tables.

Introducing Regulatory Roadmaps

These Roadmaps lay out the Government’s plans to modernize regulatory frameworks, without compromising our strong health, safety, and environmental protections. They contain proposals for legislative and regulatory amendments as well as novel regulatory approaches to accommodate emerging technologies, including the use of regulatory sandboxes and pilot projects—better aligning our regulatory frameworks with industry realities.

Budget 2019 proposes the necessary funding and legislative revisions so that regulatory departments and agencies can move forward on the Roadmaps, including providing the Canadian Food Inspection Agency, Health Canada and Transport Canada with up to $219.1 million over five years, starting in 2019–20, (with $0.5 million in remaining amortization), and $3.1 million per year on an ongoing basis.

In the coming weeks, the Government will be releasing the full Regulatory Roadmaps for each of the reviews, as well as timelines for enacting specific initiatives, which can be grouped in the following three main areas:

What Is a Regulatory Sandbox? Regulatory sandboxes are controlled “safe spaces” in which innovative products, services, business models and delivery mechanisms can be tested without immediately being subject to all of the regulatory requirements.
– European Banking Authority, 2017

Establishing a regulatory sandbox for new and innovative medical products
The regulatory approval system has not kept up with new medical technologies and processes. Health Canada proposes to modernize regulations to put in place a regulatory sandbox for new and innovative products, such as tissues developed through 3D printing, artificial intelligence, and gene therapies targeted to specific individuals. [emphasis mine]

Modernizing the regulation of clinical trials
Industry and academics have expressed concerns that regulations related to clinical trials are overly prescriptive and inconsistent. Health Canada proposes to implement a risk-based approach [emphasis mine] to clinical trials to reduce costs to industry and academics by removing unnecessary requirements for low-risk drugs and trials. The regulations will also provide the agri-food industry with the ability to carry out clinical trials within Canada on products such as food for special dietary use and novel foods.

Does the government always get 140 responses from a consultation process? Moving on, I agree with finding new approaches to regulatory processes and oversight and, by extension, new approaches to risk analysis.

Earlier in this post, I asked if someone had a budget for public relations/promotion. I wasn’t joking. My March 22, 2019 posting also included these line items in the proposed 2019 budget,

Budget 2019 proposes to make additional investments in support of the following organizations:
Stem Cell Network: Stem cell research—pioneered by two Canadians in the 1960s [James Till and Ernest McCulloch]—holds great promise for new therapies and medical treatments for respiratory and heart diseases, spinal cord injury, cancer, and many other diseases and disorders. The Stem Cell Network is a national not-for-profit organization that helps translate stem cell research into clinical applications and commercial products. To support this important work and foster Canada’s leadership in stem cell research, Budget 2019 proposes to provide the Stem Cell Network with renewed funding of $18 million over three years, starting in 2019–20.

Genome Canada: The insights derived from genomics—the study of the entire genetic information of living things encoded in their DNA and related molecules and proteins—hold the potential for breakthroughs that can improve the lives of Canadians and drive innovation and economic growth. Genome Canada is a not-for-profit organization dedicated to advancing genomics science and technology in order to create economic and social benefits for Canadians. To support Genome Canada’s operations, Budget 2019 proposes to provide Genome Canada with $100.5 million over five years, starting in 2020–21. This investment will also enable Genome Canada to launch new large-scale research competitions and projects, in collaboration with external partners, ensuring that Canada’s research community continues to have access to the resources needed to make transformative scientific breakthroughs and translate these discoveries into real-world applications.

Years ago, I managed to find a webpage with all of the proposals various organizations were submitting to a government budget committee. It was eye-opening. You can tell which organizations were able to hire someone who knew the current government buzzwords and the things that a government bureaucrat would want to hear and the organizations that didn’t.

Of course, if the government of the day is adamantly against or uninterested, no amount of persusasion will work to get your organization more money in the budget.

Finally

Reluctantly, I am inclined to explore the topic of emerging technologies such as gene-editing not only in the field of agriculture (for gene-editing of plants, fish, and animals see my November 28, 2018 posting) but also with humans. At the very least, it needs to be discussed whether we choose to participate or not.

If you are interested in the arguments against changing Canada’s prohibition against gene-editing of humans, there’s an Ocotber 2, 2017 posting on Impact Ethics by Françoise Baylis, Professor and Canada Research Chair in Bioethics and Philosophy at Dalhousie University, and Alana Cattapan, Johnson Shoyama Graduate School of Public Policy at the University of Saskatchewan, which makes some compelling arguments. Of course, it was written before the CRISPR twins (my November 28, 2018 posting).

Recaliing CRISPR Therapeutics (mentioned by Gierczak), the company received permission to run clinical trials in the US in October 2018 after the FDA (US Food and Drug Administration) lifted an earlier ban on their trials according to an Oct. 10, 2018 article by Frank Vinhuan for exome,

The partners also noted that their therapy is making progress outside of the U.S. They announced that they have received regulatory clearance in “multiple countries” to begin tests of the experimental treatment in both sickle cell disease and beta thalassemia, …

It seems to me that the quotes around “multiple countries” are meant to suggest doubt of some kind. Generally speaking, company representatives make those kinds of generalizations when they’re trying to pump up their copy. E.g., 50% increase in attendance  but no whole numbers to tell you what that means. It could mean two people attended the first year and then brought a friend the next year or 100 people attended and the next year there were 150.

Despite attempts to declare personalized medicine as having arrived, I think everything is still in flux with no preordained outcome. The future has yet to be determined but it will be and I , for one, would like to have some say in the matter.

The CRISPR ((clustered regularly interspaced short palindromic repeats)-CAS9 gene-editing technique may cause new genetic damage kerfuffle

Setting the stage

Not unexpectedly, CRISPR-Cas9  or clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 can be dangerous as these scientists note in a July 16, 2018 news item on phys.org,

Scientists at the Wellcome Sanger Institute have discovered that CRISPR/Cas9 gene editing can cause greater genetic damage in cells than was previously thought. These results create safety implications for gene therapies using CRISPR/Cas9 in the future as the unexpected damage could lead to dangerous changes in some cells.

Reported today (16 July 2018) in the journal Nature Biotechnology, the study also revealed that standard tests for detecting DNA changes miss finding this genetic damage, and that caution and specific testing will be required for any potential gene therapies.

This CRISPR-Cas9 image reminds me of popcorn,

CRISPR-associated protein Cas9 (white) from Staphylococcus aureus based on Protein Database ID 5AXW. Credit: Thomas Splettstoesser (Wikipedia, CC BY-SA 4.0)[ downloaded from https://phys.org/news/2018-07-genome-crisprcas9-gene-higher-thought.html#jCp]

A July 16, 2018 Wellcome Sanger Institute press release (also on EurekAlert), which originated the news item, offers a little more explanation,

CRISPR/Cas9 is one of the newest genome editing tools. It can alter sections of DNA in cells by cutting at specific points and introducing changes at that location. Already extensively used in scientific research, CRISPR/Cas9 has also been seen as a promising way to create potential genome editing treatments for diseases such as HIV, cancer or sickle cell disease. Such therapeutics could inactivate a disease-causing gene, or correct a genetic mutation. However, any potential treatments would have to prove that they were safe.

Previous research had not shown many unforeseen mutations from CRISPR/Cas9 in the DNA at the genome editing target site. To investigate this further the researchers carried out a full systematic study in both mouse and human cells and discovered that CRISPR/Cas9 frequently caused extensive mutations, but at a greater distance from the target site.

The researchers found many of the cells had large genetic rearrangements such as DNA deletions and insertions. These could lead to important genes being switched on or off, which could have major implications for CRISPR/Cas9 use in therapies. In addition, some of these changes were too far away from the target site to be seen with standard genotyping methods.

Prof Allan Bradley, corresponding author on the study from the Wellcome Sanger Institute, said: “This is the first systematic assessment of unexpected events resulting from CRISPR/Cas9 editing in therapeutically relevant cells, and we found that changes in the DNA have been seriously underestimated before now. It is important that anyone thinking of using this technology for gene therapy proceeds with caution, and looks very carefully to check for possible harmful effects.”

Michael Kosicki, the first author from the Wellcome Sanger Institute, said: “My initial experiment used CRISPR/Cas9 as a tool to study gene activity, however it became clear that something unexpected was happening. Once we realised the extent of the genetic rearrangements we studied it systematically, looking at different genes and different therapeutically relevant cell lines, and showed that the CRISPR/Cas9 effects held true.”

The work has implications for how CRISPR/Cas9 is used therapeutically and is likely to re-spark researchers’ interest in finding alternatives to the standard CRISPR/Cas9 method for gene editing.

Prof Maria Jasin, an independent researcher from Memorial Slone Kettering Cancer Centre, New York, who was not involved in the study said: “This study is the first to assess the repertoire of genomic damage arising at a CRISPR/Cas9 cleavage site. While it is not known if genomic sites in other cell lines will be affected in the same way, this study shows that further research and specific testing is needed before CRISPR/Cas9 is used clinically.”

For anyone who’d like to better understand the terms gene editing and CRISPR-Cas9, the Wellcome Sanger Institute provides these explanatory webpages, What is genome editing? and What is CRISPR-Cas9?

For the more advanced, here’s a link and a citation for the paper,

Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements by Michael Kosicki, Kärt Tomberg, & Allan Bradley. Nature Biotechnology DOI: https://doi.org/10.1038/nbt.4192 Published 16 July 2018

This paper appears to be open access.

The kerfuffle

It seems this news has affected the CRISPR market. From a July 16, 2018 article by Cale Guthrie Weissman for Fast Company,

… CRISPR could unknowingly delete or alter non-targeted genes, which could lead to myriad unintended consequences. This is especially frightening, since the technology is going to be used in human clinical trials.

Meanwhile, other scientists working with CRISPR are trying to downplay the findings, telling STAT [a life sciences and business journalism website] that there have been no reported adverse effects similar to what the study describes. The news, however, has brought about a market reaction–at least three publicly traded companies that focus on CRISPR-based therapies are in stock nosedive. Crispr Therapeutics is down by over 6%; Editas fell by over 3%; and Intellia Therapeutics dropped by over 5%. [emphasis mine]

Damage control

Gaetan Burgio (geneticist, Australian National University)  in a July 16, 2018 essay on phys.org (originating from The Conversation) suggests some calm (Note: Links have been removed),

But a new study has called into question the precision of the technique [CRISPR gene editing technology].

The hope for gene editing is that it will be able to cure and correct diseases. To date, many successes have been reported, including curing deafness in mice, and in altering cells to cure cancer.

Some 17 clinical trials in human patients are registered [emphasis mine] testing gene editing on leukaemias, brain cancers and sickle cell anaemia (where red blood cells are misshaped, causing them to die). Before implementing CRISPR technology in clinics to treat cancer or congenital disorders, we must address whether the technique is safe and accurate.

There are a few options for getting around this problem. One option is to isolate the cells we wish to edit from the body and reinject only the ones we know have been correctly edited.

For example, lymphocytes (white blood cells) that are crucial to killing cancer cells could be taken out of the body, then modified using CRISPR to heighten their cancer-killing properties. The DNA of these cells could be sequenced in detail, and only the cells accurately and specifically gene-modified would be selected and delivered back into the body to kill the cancer cells.

While this strategy is valid for cells we can isolate from the body, some cells, such as neurons and muscles, cannot be removed from the body. These types of cells might not be suitable for gene editing using Cas9 scissors.

Fortunately, researchers have discovered other forms of CRISPR systems that don’t require the DNA to be cut. Some CRISPR systems only cut the RNA, not the DNA (DNA contains genetic instructions, RNA convey the instructions on how to synthesise proteins).

As RNA [ribonucleic acid] remains in our cells only for a specific period of time before being degraded, this would allow us to control the timing and duration of the CRISPR system delivery and reverse it (so the scissors are only functional for a short period of time).

This was found to be successful for dementia in mice. Similarly, some CRISPR systems simply change the letters of the DNA, rather than cutting them. This was successful for specific mutations causing diseases such as hereditary deafness in mice.

I agree with Burgio’s conclusion (not included here) that we have a lot more to learn and I can’t help wondering why there are 17 registered human clinical trials at this point.

Nanoparticle-based delivery platform for CRISPR-Cas9 (gene-editing technology)

A February 18, 2018 King Abdullah University of Science and Technology (KAUST; Saudi Arabia) news release (also on EurekAlert but published on Feb. 20, 2018) describes a new technology for delivering CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 into cells,

A new delivery system for introducing gene-editing technology into cells could help safely and efficiently correct disease-causing mutations in patients.

The system, developed by KAUST scientists, is the first to use sponge-like ensembles of metal ions and organic molecules to coat the molecular components of the precision DNA-editing technology known as CRISPR/Cas9, allowing efficient release of the genome-editing machinery inside the cell.

“This method presents an easy and economically feasible route to improve on the delivery problems that accompany RNA-based therapeutic approaches,” says Niveen Khashab, the associate professor of chemical sciences at KAUST who led the study. “This may permit such formulations to be eventually used for treating genetic diseases effectively in the future.”

CRISPR/Cas9 has a double delivery problem: For the gene-editing technology to work like a molecular Swiss Army knife, both a large protein (the Cas9 cutting enzyme) and a highly charged RNA component (the guide RNA used for DNA targeting) must each get from the outside of the cell into the cytoplasm and finally into the nucleus, all without getting trapped in the tiny intracellular bubbles that are known as endosomes.

To solve this problem, Khashab and her lab turned to a nano-sized type of porous material known as a zeolitic imidazolate framework, which forms a cage-like structure into which other molecules can be placed. The researchers encapsulated the Cas9 protein and guide RNA in this material and then introduced the resulting nanoparticles into hamster cells.

The encapsulated CRISPR-Cas9 constructs were not toxic to the cells. And because particles in the coating material become positively charged when absorbed into endosomes, they caused these membrane-bound bubbles to burst, freeing the CRISPR-Cas9 machinery to travel to the nucleus, home to the cell’s genome. There the gene-editing technology could get to work.

Using a guide RNA designed to target a gene that caused the cells to glow green under fluorescent light, Khashab and her team showed that they could reduce the expression of this gene by 37 percent over four days with their technology. “These cage-like structures are biocompatible and can be triggered on demand, making them smart options to overcome delivery problems of genetic materials and proteins,” says the study’s first author Shahad Alsaiari, a Ph.D. student in Khashab’s lab.

The researchers’ plan to test their system in human cells and in mice, and eventually, they hope, in clinical trials.

The zeolitic imidazolate framework forms a cage-like scaffold over the CRISPR/Cas9 machinery.. Reprinted (adapted) with permission from Alsaiari, S.K., Patil, S., Alyami, M., Alamoudi, K.O., Aleisa, F.A., Merzaban, J., Li M. & Khashab, N.M. Endosomal escape and delivery of CRISPR/Cas9 genome editing machinery enabled by nanoscale zeolitic imidazolate framework. Journal of the American Chemical Society 140, 143–146 (2018). © 2018 American Chemical Society; KAUST Xavier Pita and Heno Huang ][downloaded from https://discovery.kaust.edu.sa/en/article/475/a%250adelivery-platform-for-gene-editing-technology]

Here’s a link to and a citation for the paper,

Endosomal Escape and Delivery of CRISPR/Cas9 Genome Editing Machinery Enabled by Nanoscale Zeolitic Imidazolate Framework by Shahad K. Alsaiari, Sachin Patil, Mram Alyami, Kholod O. Alamoudi, Fajr A. Aleisa, Jasmeen S. Merzaban, Mo Li, and Niveen M. Khashab. J. Am. Chem. Soc., 2018, 140 (1), pp 143–146 DOI: 10.1021/jacs.7b11754 Publication Date (Web): December 22, 2017

Copyright © 2017 American Chemical Society

This paper is behind a paywall.

CRISPR-Cas12a as a new diagnostic tool

Similar to Cas9, Cas12a is has an added feature as noted in this February 15, 2018 news item on ScienceDaily,

Utilizing an unsuspected activity of the CRISPR-Cas12a protein, researchers created a simple diagnostic system called DETECTR to analyze cells, blood, saliva, urine and stool to detect genetic mutations, cancer and antibiotic resistance and also diagnose bacterial and viral infections. The scientists discovered that when Cas12a binds its double-stranded DNA target, it indiscriminately chews up all single-stranded DNA. They then created reporter molecules attached to single-stranded DNA to signal when Cas12a finds its target.

A February 15, 2018 University of California at Berkeley (UC Berkeley) news release by Robert Sanders and which originated the news item, provides more detail and history,

CRISPR-Cas12a, one of the DNA-cutting proteins revolutionizing biology today, has an unexpected side effect that makes it an ideal enzyme for simple, rapid and accurate disease diagnostics.

blood in test tube

(iStock)

Cas12a, discovered in 2015 and originally called Cpf1, is like the well-known Cas9 protein that UC Berkeley’s Jennifer Doudna and colleague Emmanuelle Charpentier turned into a powerful gene-editing tool in 2012.

CRISPR-Cas9 has supercharged biological research in a mere six years, speeding up exploration of the causes of disease and sparking many potential new therapies. Cas12a was a major addition to the gene-cutting toolbox, able to cut double-stranded DNA at places that Cas9 can’t, and, because it leaves ragged edges, perhaps easier to use when inserting a new gene at the DNA cut.

But co-first authors Janice Chen, Enbo Ma and Lucas Harrington in Doudna’s lab discovered that when Cas12a binds and cuts a targeted double-stranded DNA sequence, it unexpectedly unleashes indiscriminate cutting of all single-stranded DNA in a test tube.

Most of the DNA in a cell is in the form of a double-stranded helix, so this is not necessarily a problem for gene-editing applications. But it does allow researchers to use a single-stranded “reporter” molecule with the CRISPR-Cas12a protein, which produces an unambiguous fluorescent signal when Cas12a has found its target.

“We continue to be fascinated by the functions of bacterial CRISPR systems and how mechanistic understanding leads to opportunities for new technologies,” said Doudna, a professor of molecular and cell biology and of chemistry and a Howard Hughes Medical Institute investigator.

DETECTR diagnostics

The new DETECTR system based on CRISPR-Cas12a can analyze cells, blood, saliva, urine and stool to detect genetic mutations, cancer and antibiotic resistance as well as diagnose bacterial and viral infections. Target DNA is amplified by RPA to make it easier for Cas12a to find it and bind, unleashing indiscriminate cutting of single-stranded DNA, including DNA attached to a fluorescent marker (gold star) that tells researchers that Cas12a has found its target.

The UC Berkeley researchers, along with their colleagues at UC San Francisco, will publish their findings Feb. 15 [2018] via the journal Science’s fast-track service, First Release.

The researchers developed a diagnostic system they dubbed the DNA Endonuclease Targeted CRISPR Trans Reporter, or DETECTR, for quick and easy point-of-care detection of even small amounts of DNA in clinical samples. It involves adding all reagents in a single reaction: CRISPR-Cas12a and its RNA targeting sequence (guide RNA), fluorescent reporter molecule and an isothermal amplification system called recombinase polymerase amplification (RPA), which is similar to polymerase chain reaction (PCR). When warmed to body temperature, RPA rapidly multiplies the number of copies of the target DNA, boosting the chances Cas12a will find one of them, bind and unleash single-strand DNA cutting, resulting in a fluorescent readout.

The UC Berkeley researchers tested this strategy using patient samples containing human papilloma virus (HPV), in collaboration with Joel Palefsky’s lab at UC San Francisco. Using DETECTR, they were able to demonstrate accurate detection of the “high-risk” HPV types 16 and 18 in samples infected with many different HPV types.

“This protein works as a robust tool to detect DNA from a variety of sources,” Chen said. “We want to push the limits of the technology, which is potentially applicable in any point-of-care diagnostic situation where there is a DNA component, including cancer and infectious disease.”

The indiscriminate cutting of all single-stranded DNA, which the researchers discovered holds true for all related Cas12 molecules, but not Cas9, may have unwanted effects in genome editing applications, but more research is needed on this topic, Chen said. During the transcription of genes, for example, the cell briefly creates single strands of DNA that could accidentally be cut by Cas12a.

The activity of the Cas12 proteins is similar to that of another family of CRISPR enzymes, Cas13a, which chew up RNA after binding to a target RNA sequence. Various teams, including Doudna’s, are developing diagnostic tests using Cas13a that could, for example, detect the RNA genome of HIV.

infographic about DETECTR system

(Infographic by the Howard Hughes Medical Institute)

These new tools have been repurposed from their original role in microbes where they serve as adaptive immune systems to fend off viral infections. In these bacteria, Cas proteins store records of past infections and use these “memories” to identify harmful DNA during infections. Cas12a, the protein used in this study, then cuts the invading DNA, saving the bacteria from being taken over by the virus.

The chance discovery of Cas12a’s unusual behavior highlights the importance of basic research, Chen said, since it came from a basic curiosity about the mechanism Cas12a uses to cleave double-stranded DNA.

“It’s cool that, by going after the question of the cleavage mechanism of this protein, we uncovered what we think is a very powerful technology useful in an array of applications,” Chen said.

Here’s a link to and a citation for the paper,

CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity by Janice S. Chen, Enbo Ma, Lucas B. Harrington, Maria Da Costa, Xinran Tian, Joel M. Palefsky, Jennifer A. Doudna. Science 15 Feb 2018: eaar6245 DOI: 10.1126/science.aar6245

This paper is behind a paywall.