Tag Archives: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

Even a ‘good’ gene edit can go wrong

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An October 24, 2022 news item on ScienceDaily highlights research into better understanding problems with ‘good’ CRISPR (clustered regularly interspaced short palindromic repeats) gene editing,

A Rice University lab is leading the effort to reveal potential threats to the efficacy and safety of therapies based on CRISPR-Cas9, the Nobel Prize-winning gene editing technique, even when it appears to be working as planned.

Bioengineer Gang Bao of Rice’s George R. Brown School of Engineering and his team point out in a paper published in Science Advances that while off-target edits to DNA have long been a cause for concern, unseen changes that accompany on-target edits also need to be recognized — and quantified.

Bao noted a 2018 Nature Biotechnology paper indicated the presence of large deletions. “That’s when we started looking into what we can do to quantify them, due to CRISPR-Cas9 systems designed for treating sickle cell disease,” he said.

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An October 24, 2022 Rice University news release (also on EurekAlert), which originated the news item, details the concerns (Note: Links have been removed),

Bao has been a strong proponent of CRISPR-Cas9 as a tool to treat sickle cell disease, a quest that has brought him and his colleagues ever closer to a cure. Now the researchers fear that large deletions or other undetected changes due to gene editing could persist in stem cells as they divide and differentiate, thus have long-term implications for health.

“We do not have a good understanding of why a few thousand bases of DNA at the Cas9 cut site can go missing and the DNA double-strand breaks can still be rejoined efficiently,” Bao said. “That’s the first question, and we have some hypotheses. The second is, what are the biological consequences? Large deletions (LDs) can reach to nearby genes and disrupt the expression of both the target gene and the nearby genes. It is unclear if LDs could result in the expression of truncated proteins. 

“You could also have proteins that misfold, or proteins with an extra domain because of large insertions,” he said. “All kinds of things could happen, and the cells could die or have abnormal functions.”

His lab developed a procedure that uses single-molecule, real-time (SMRT) sequencing with dual unique molecular identifiers (UMI) to find and quantify unintended LDs along with large insertions and local chromosomal rearrangements that accompany small insertions/deletions (INDELs) at a Cas9 on-target cut site. 

“To quantify large gene modifications, we need to perform long-range PCR, but that could induce artifacts during DNA amplification,” Bao said. “So we used UMIs of 18 bases as a kind of barcode.”

“We add them to the DNA molecules we want to amplify to identify specific DNA molecules as a way to reduce or eliminate artifacts due to long-range PCR,” he said. “We also developed a bioinformatics pipeline to analyze SMRT sequencing data and quantified the LDs and large insertions.”

The Bao lab’s tool, called LongAmp-seq (for long-amplicon sequencing), accurately quantifies both small INDELs and large LDs. Unlike SMRT-seq, which requires the use of a long-read sequencer often only available at a core facility, LongAmp-seq can be performed using a short-read sequencer.

To test the strategy, the lab team led by Rice alumna Julie Park, now an assistant research professor of bioengineering, used Streptococcus pyogenes Cas9 to edit beta-globin (HBB), gamma-globin (HBG) and B-cell lymphoma/leukemia 11A (BCL11A) enhancers in hematopoietic stem and progenitor cells (HSPC) from patients with sickle cell disease, and the PD-1 gene in primary T-cells.  

They found large deletions of up to several thousand bases occurred at high frequency in HSPCs: up to 35.4% in HBB, 14.3% in HBG and 15.2% in BCL11A genes, as well as on the PD-1 (15.2%) gene in T-cells. 

Since two of the specific CRISPR guide RNAs tested by the Bao lab are being used in clinical trials to treat sickle cell disease, he said it’s important to determine the biological consequences of large gene modifications due to Cas9-induced double-strand breaks. 

Bao said the Rice team is currently looking downstream to analyze the consequences of long deletions on messenger RNA, the mediator that carries code for ribosomes to make proteins. “Then we’ll move on to the protein level,” Bao said. “We want to know if these large deletions and insertions persist after the gene-edited HSPCs are transplantation into mice and patients.”  

Co-authors of the study from Rice are graduate students Mingming Cao and Yilei Fu, alumni Yidan Pan and Timothy Davis, research specialist Lavanya Saxena, microscopist/bioinstrumentation specialist Harshavardhan Deshmukh and Todd Treangen, an assistant professor of computer science, and Emory University’s Vivien Sheehan, an associate professor of pediatrics. 

Bao is the department chair and Foyt Family Professor of Bioengineering, a professor of chemistry, materials science and nanoengineering, and mechanical engineering, and a CPRIT Scholar in Cancer Research.

The National Institutes of Health (R01HL152314, OT2HL154977) supported the research.

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

Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR-Cas9 gene editing by So Hyun Park, Mingming Cao, Yidan Pan, Timothy H. Davis, Lavanya Saxena, Harshavardhan Deshmukh, Yilei Fu, Todd Treangen, Vivien A. Sheehan, and Gang Bao. Science Advances Vol 8, Issue 42 DOI: 10.1126/sciadv.abo7676 First published online: 21 Oct 2022 Published in print: March 3, 2023

This paper is behind a paywall.

A CRISPR (clustered regularly interspaced short palindromic repeats) anniversary

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June 2022 was the 10th anniversary of the publication of a study the paved the way for CRISPR-Cas9 gene editing and Sophie Fessl’s June 28, 2022 article for The Scientist offers a brief history (Note: Links have been removed),

Ten years ago, Emmanuelle Charpentier and Jennifer Doudna published the study that paved the way for a new kind of genome editing: the suite of technologies now known as CRISPR. Writing in [the journal] Science, they adapted an RNA-mediated bacterial immune defense into a targeted DNA-altering system. “Our study . . . highlights the potential to exploit the system for RNA-programmable genome editing,” they conclude in the abstract of their paper—a potential that, in the intervening years, transformed the life sciences. 

From gene drives to screens, and diagnostics to therapeutics, CRISPR nucleic acids and the Cas enzymes with which they’re frequently paired have revolutionized how scientists tinker with DNA and RNA. … altering the code of life with CRISPR has been marred by ethical concerns. Perhaps the most prominent example was when Chinese scientist He Jiankui created the first gene edited babies using CRISPR/Cas9 genome editing. Doudna condemned Jiankui’s work, for which he was jailed, as “risky and medically unnecessary” and a “shocking reminder of the scientific and ethical challenges raised by this powerful technology.” 

There’s also the fact that legal battles over who gets to claim ownership of the system’s many applications have persisted almost as long as the technology has been around. Both Doudna and Charpentier’s teams from the University of California, Berkeley, and the University of Vienna and a team led by the Broad Institute’s Feng Zhang claim to be the first to have adapted CRISPR-Cas9 for gene editing in complex cells (eukaryotes). Patent offices in different countries have reached varying decisions, but in the US, the latest rulings say that the Broad Institute of MIT [Massachusetts Institute of Technology] and Harvard retains intellectual property of using CRISPR-Cas9 in eukaryotes, while Emmanuelle Charpentier, the University of California, and the University of Vienna maintain their original patent over using CRISPR-Cas9 for editing in vitro and in prokaryotes. 

Still, despite the controversies, the technique continues to be explored academically and commercially for everything from gene therapy to crop improvement. Here’s a look at seven different ways scientists have utilized CRISPR.

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Fessl goes on to give a brief overview of CRISPR and gene drives, genetic screens, diagnostics, including COVID-19 tests, gene therapy, therapeutics, crop and livestock improvement, and basic research.

For anyone interested in the ethical issues (with an in depth look at the Dr. He Jiankui story), I suggest reading either or both Eben Kirksey’s 2020 book, “The Mutant Project; Inside the Global Race to Genetically Modify Humans,”

An anthropologist visits the frontiers of genetics, medicine, and technology to ask: Whose values are guiding gene editing experiments? And what does this new era of scientific inquiry mean for the future of the human species?

“That rare kind of scholarship that is also a page-turner.”
—Britt Wray, author of Rise of the Necrofauna

At a conference in Hong Kong in November 2018, Dr. He Jiankui announced that he had created the first genetically modified babies—twin girls named Lulu and Nana—sending shockwaves around the world. A year later, a Chinese court sentenced Dr. He to three years in prison for “illegal medical practice.”

As scientists elsewhere start to catch up with China’s vast genetic research program, gene editing is fueling an innovation economy that threatens to widen racial and economic inequality. Fundamental questions about science, health, and social justice are at stake: Who gets access to gene editing technologies? As countries loosen regulations around the globe, from the U.S. to Indonesia, can we shape research agendas to promote an ethical and fair society?

Eben Kirksey takes us on a groundbreaking journey to meet the key scientists, lobbyists, and entrepreneurs who are bringing cutting-edge genetic engineering tools like CRISPR—created by Nobel Prize-winning biochemists Jennifer Doudna and Emmanuelle Charpentier—to your local clinic. He also ventures beyond the scientific echo chamber, talking to disabled scholars, doctors, hackers, chronically-ill patients, and activists who have alternative visions of a genetically modified future for humanity.

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and/or Kevin Davies’s 2020 book, “Editing Humanity: The CRISPR Revolution and the New Era of Genome Editing,”

One of the world’s leading experts on genetics unravels one of the most important breakthroughs in modern science and medicine. 

If our genes are, to a great extent, our destiny, then what would happen if mankind could engineer and alter the very essence of our DNA coding? Millions might be spared the devastating effects of hereditary disease or the challenges of disability, whether it was the pain of sickle-cell anemia to the ravages of Huntington’s disease.

But this power to “play God” also raises major ethical questions and poses threats for potential misuse. For decades, these questions have lived exclusively in the realm of science fiction, but as Kevin Davies powerfully reveals in his new book, this is all about to change.

Engrossing and page-turning, Editing Humanity takes readers inside the fascinating world of a new gene editing technology called CRISPR, a high-powered genetic toolkit that enables scientists to not only engineer but to edit the DNA of any organism down to the individual building blocks of the genetic code.

Davies introduces readers to arguably the most profound scientific breakthrough of our time. He tracks the scientists on the front lines of its research to the patients whose powerful stories bring the narrative movingly to human scale.

Though the birth of the “CRISPR babies” in China made international news, there is much more to the story of CRISPR than headlines seemingly ripped from science fiction. In Editing Humanity, Davies sheds light on the implications that this new technology can have on our everyday lives and in the lives of generations to come.

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Kevin Davies is the executive editor of The CRISPR Journal and the founding editor of Nature Genetics. He holds an MA in biochemistry from the University of Oxford and a PhD in molecular genetics from the University of London. He is the author of Cracking the Genome, The $1,000 Genome, and co-authored a new edition of DNA: The Story of the Genetic Revolution with Nobel Laureate James D. Watson and Andrew Berry. In 2017, Kevin was selected for a Guggenheim Fellowship in science writing.

I’ve read both books and while some of the same ground is covered, the perspectives diverge somewhat. Both authors offer a more nuanced discussion of the issues than was the case in the original reporting about Dr. He’s work.

What is CRISPRnano?

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To answer the question, CRISPRnano is a computational webserver for identifying gene-edited cells. (For those unfamiliar with CRISPR, it stands for clustered regularly interspaced short palindromic repeats’ a form of gene editing,)

The webserver was announced in a July 15, 2022 news item on Nanowerk but first, there’s an explanation of why this server is needed,

Diseases of genetic cause can be investigated by inducing the respective mutations in cell lines that are then used to model human diseases. The overall aim is to elucidate underlying mechanisms, interactions with environmental factors and ideally to find curative strategies.

A crucial step in generating genetically modified cell models is to verify the inserted mutation. Therefore, the genetic information carrier of the cells is decoded (sequencing) and compared to the reference set of genetic information in healthy individuals (genotyping).

To support scientists with the comparison, different workflows and software are available, but many of them require expensive high-tier sequencers or manual curation efforts.

A July 15, 2022 IUF – Leibniz-Institut für umweltmedizinische Forschung (Leibniz Research Institute for Environmental Medicine) press release, which originated the news item, includes details about the server,

To address this issue, a team of scientists from the Genome Engineering and Model Development lab at the IUF – Leibniz Research Institute for Environmental Medicine in Düsseldorf, led by Dr. Andrea Rossi, developed a robust, versatile, and easy-to-use computational webserver named CRISPRnano (https://www.crisprnano.de/) that enables the analysis of noisy reads generated by affordable and portable sequencers including Oxford Nanopore Technologies (ONT) devices. CRISPRnano allows fast and accurate identification, quantification, and visualization of genetically modified cell lines, it is compatible with Next Generation Sequencing (NGS) and ONT sequencing reads, and it can be used without an internet connection. The according study was published in the renowned scientific journal Nucleic Acids Research.

Here’s a link to and a citation for the related study,

Identification of genome edited cells using CRISPRnano by Thach Nguyen, Haribaskar Ramachandran, Soraia Martins, Jean Krutmann, Andrea Rossi. Nucleic Acids Research, Volume 50, Issue W1, 5 July 2022, Pages W199–W203, DOI: https://doi.org/10.1093/nar/gkac440 Published: 30 May 2022

This paper appears to be open access.

Use Gene Editing to Make Better Babies (a February 17, 2022 livestreamed debate from 05:00 PM − 06:30 PM EST)

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I have high hopes for this debate on gene edited babies. Intelligence Squared US convenes good debates. (I watched their ‘de-extinction’ debate back in 2019, which coincidentally, featured George Church, one of the debaters in this event.) Not ‘good’ in that I necessarily agree or am interested in the topics but good as in thoughtful. Here’s more from the organization’s mission on their What is IQ2US? webpage,

A nonpartisan, nonprofit organization, Intelligence Squared U.S. addresses a fundamental problem in America: the extreme polarization of our nation and our politics.

Our mission is to restore critical thinking, facts, reason, and civility to American public discourse.

More about the upcoming debate can be found on the Use Gene Editing to Make Better Babies event page,

Use Gene Editing to Make Better Babies
Hosted By John Donvan

Thursday, February 17, 2022
05:00 PM − 06:30 PM EST

A genetic disease runs in your family. Your doctor tells you that, should you wish to have a child, that child is likely to also carry the disease. But a new gene-editing technology could change your fate. It could ensure that your baby is — and remains — healthy. Even more, it could potentially make sure your grandchildren are also free of the disease. What do you do? Now, imagine it’s not a rare genetic disorder, but general illness, or eye color, or cognitive ability, or athleticism. Do you opt into this new world of genetically edited humans? And what if it’s not just you. What your friends, neighbors, and colleagues are also embracing this genetic revolution? Right now, science doesn’t give you that choice. But huge advancements in CRISPR [clustered regularly interspaced short palindromic repeats] technology are making human gene editing a reality. In fact, in 2018, a Chinese scientist announced the first genetically modified babies; twin girls made to resist HIV, smallpox, and malaria. The promise of this technology is clear. But gene editing is not without its perils. Its critics say the technology is destined to exacerbate inequality, pressure all parents (and nations) into editing their children to stay competitive, and meddling with the most basic aspect of our humanity. In this context, we ask the question: Should we use gene editing to make better babies?

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Main Points

The use of gene editing allows for couples to have children when they might otherwise have that option unavailable for them. It also allows for less to be left to chance during the pregnancy.

Gene editing will allow for babies to be born with reduced or eliminated chances of inheriting and passing on genes linked to diseases. We have a moral imperative to use technology that will improve the quality of life.

It is only a matter of time before gene editing becomes a widespread technology, potentially used by competitors and rivals on the international stage. If we have the technology, we should use it to our advantage to remain competitive.

The use of gene editing to create “better” outcomes in children will inherently create social stratification based on any gene editing, likely reflecting existing socioeconomic status. Additionally, the term ‘better’ is arbitrary and potentially short-sighted and dangerous.

Currently, there exist reasonable alternatives to gene editing for every condition for which gene editing can be used. 

The technology is still developing, and the long-term effects of any gene-editing could be potentially dangerous with consequences echoing throughout the gene environment. 

A February 8, 2022 Intelligence Squared U.S. news release about the upcoming debate (received via email) provides details about the debaters,

FOR THE MOTION – BIOS

* George Church, Geneticist & Founder, Personal Genome Project 
George Church is one of the nation’s leading geneticists and scholars. He is a professor of genetics at Harvard Medical School and MIT. In 1984, he developed the first direct genomic sequencing method, which resulted in the first genome sequence. He also helped initiate the Human Genome Project in 1984 and the Personal Genome Project in 2005. Church also serves as the director of the National Institutes of Health Center of Excellence in Genomic Science.  

* Amy Webb, Futurist & Author, “The Genesis Machine”  
Amy Webb is an award-winning author and futurist. She is the founder and CEO of the Future Today Institute and was named one of five women changing the world by Forbes. Her new book, “The Genesis Machine,” explores the future of synthetic biology, including human gene editing. Webb is a professor of strategic foresight at New York University’s Stern School of Business and has been elected a life member of the Council on Foreign Relations.  

AGAINST THE MOTION – BIOS

* Marcy Darnovsky, Policy Advocate & Executive Director, Center for Genetics and Society 
Marcy Darnovsky is a policy advocate and one of the most prominent voices on the politics of human biotechnology. As executive director of the Center for Genetics and Society, Darnovsky is focused on the social justice and public interest implications of gene editing. This work is informed by her background as an organizer and advocate in a range of environmental and progressive political movements.    

* Françoise Baylis, Philosopher & Author, “Altered Inheritance”  
Françoise Baylis is a philosopher whose innovative work in bioethics, at the intersection of policy and practice, has stretched the very boundaries of the field. She is the author of “Altered Inheritance: CRISPR and the Ethics of Human Genome Editing,” which explores the scientific, ethical, and political implications of human genome editing. Baylis is a research professor at Dalhousie University and a fellow of the Canadian Academy of Health Sciences. In 2017, she was awarded the Canadian Bioethics Society Lifetime Achievement Award. 

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Getting back to the Use Gene Editing to Make Better Babies event page, there are a few options,

Request a Ticket

Have a question? Ask us

There’s also an option to Vote For or Against the Motion but you’ll have to go to the Use Gene Editing to Make Better Babies event page.

Two of the debaters have been mentioned on this blog before, George Church and Françoise Baylis. There are several references to Church including this mention with regard to Dr. He Jiankui and his CRISPR twins (July 28, 2020 posting). Françoise Baylis features in four 2019 postings with the most recent being this October 17, 2019 piece.

For anyone curious about the ‘de-extinction’ debate, it was described here in a January 18, 2019 posting prior to the event.

World CRISPR Day on October 20, 2021 from 8:00 a.m. – 6:00 p.m. PDT

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H/t to rapper Baba Brinkman (born in Canada and based in New York City) for the tweet/retweet about his upcoming appearance at World CRISPR (clustered regularly interspaced palindromic repeats) Day on October 20, 2021 from 8:00 a.m. – 6:00 p.m. PDT,

Baba Brinkman @BabaBrinkman

True facts! I’ve been working with incredible #CRISPR innovator @Synthego and the @EventRapInc team, and tomorrow is #WorldCRISPRDay! Look for new DNA-themed videos and streamed performances all day from @HilaTheKilla, @CoreyJGray, @ZEPS, @MCAbdominal and me. Sign up to watch!

Synthego
@Synthego· 2h
Multiple musical notes BREAKING NEWS Multiple musical notes We’re delighted to announce that @BabaBrinkman will be performing live at #WorldCRISPRDay! Register today so you don’t miss out on this special and exclusive performance at the biggest event in #CRISPR! https://hubs.li/H0ZGfSG0

World CRISPR Day (it’s free) is being hosted by Synthego, from their About Us (company) webpage,

Synthego is a genome engineering company that enables the acceleration of life science research and development in the pursuit of improved human health.

The company leverages machine learning, automation, and gene editing to build platforms for science at scale. With its foundations in engineering disciplines, the company’s platform technologies vertically integrate proprietary hardware, software, bioinformatics, chemistries, and molecular biology to advance basic research, target validation, and clinical trials.

With its technologies cited in hundreds of peer-reviewed publications and utilized by thousands of commercial and academic researchers and therapeutic drug developers, Synthego is at the forefront of innovation enabling the next generation of medicines by delivering genome editing at an unprecedented scale.

Here’s the company’s (undated) announcement about the upcoming World CRISPR Day,

Synthego is proud to host the 2nd annual World CRISPR Day virtual event on October 20, 2021, where we can share, listen, and learn about the latest advancements in CRISPR. The day will include presentations from the world’s leading Genome Engineers, a panel discussion featuring the women of CRISPR, and much more! Don’t miss your chance to learn from the experts how CRISPR is editing the future of medicine.

Despite the COVID-related challenges that the global research community continues to face, scientists have persevered in their relentless pursuit of advancing human health. The field of CRISPR has been no exception. With development of new CRISPR innovations, drug discovery and diagnostic methods, and numerous successful reports of CRISPR-based cell and gene therapy clinical trials, the promise of CRISPR in the clinic is becoming a reality.

Join us at World CRISPR Day to hear academic and industry experts talk about their transformative research, visit our partner’s booths, take advantage of the different networking sessions with your peers, and much more!

Register now for free!

You can find World CRISPR Day 2021 here and you can find Baba Brinkman’s website here.

Having looked at the pop up pages describing the panel discussions and participants and having looked at their World CRISPR Day 2021 and 2020 videos, I strongly suspect that this day focuses on CRISPR as the solution to any number of problems in the life sciences, an area, where coincidentally, Synthego and its partners have significant expertise. With that proviso in mind, I’m sure this will be a very interesting and worthwhile day.

Five country survey of reactions to food genome editing

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Weirdly and even though most of this paper’s authors are from the University of British Columbia (UBC; Canada), only one press release was issued and that was by the lead author’s (Gesa Busch) home institution, the University of Göttingen (Germany).

I’m glad Busch, the other authors, and the work are getting some attention (if not as much as I think they should).

From a July 9, 2021 University of Göttingen press release (also on EurekAlert but published on July 12, 2021),

A research team from the University of Göttingen and the University of British Columbia (Canada) has investigated how people in five different countries react to various usages of genome editing in agriculture. The researchers looked at which uses are accepted and how the risks and benefits of the new breeding technologies are rated by people. The results show only minor differences between the countries studied – Germany, Italy, Canada, Austria and the USA. In all countries, making changes to the genome is more likely to be deemed acceptable when used in crops rather than in livestock. The study was published in Agriculture and Human Values.

Relatively new breeding technologies, such as CRISPR [clustered regularly interspaced short palindromic repeats) gene editing, have enabled a range of new opportunities for plant and animal breeding. In the EU, the technology falls under genetic engineering legislation and is therefore subject to rigorous restrictions. However, the use of gene technologies remains controversial. Between June and November 2019, the research team collected views on this topic via online surveys from around 3,700 people from five countries. Five different applications of gene editing were evaluated: three relate to disease resistance in people, plants, or animals; and two relate to achieving either better quality of produce or a larger quantity of product from cattle.

“We were able to observe that the purpose of the gene modification plays a major role in how it is rated,” says first author Dr Gesa Busch from the University of Göttingen. “If the technology is used to make animals resistant to disease, approval is greater than if the technology is used to increase the output from animals.” Overall, however, the respondents reacted very differently to the uses of the new breeding methods. Four different groups can be identified: strong supporters, supporters, neutrals, and opponents of the technology. The opponents (24 per cent) identify high risks and calls for a ban of the technology, regardless of possible benefits. The strong supporters (21 per cent) see few risks and many advantages. The supporters (26 per cent) see many advantages but also risks. Whereas those who were neutral (29 per cent) show no strong opinion on the subject.

This study was made possible through funding from the Free University of Bozen-Bolzano and Genome BC.

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

Citizen views on genome editing: effects of species and purpose by Gesa Busch, Erin Ryan, Marina A. G. von Keyserlingk & Daniel M. Weary. Agriculture and Human Values (2021) Published: DOI: https://doi.org/10.1007/s10460-021-10235-9

This paper is open access.

Methodology

I have one quick comment about the methodology. It can be difficult to get a sample that breaks down along demographic lines that is close to or identical to national statistics. That said, it was striking to me that every country was under represented in the ’60 years+ ‘ category. In Canada, it was by 10 percentage points (roughly). For other countries the point spread was significantly wider. In Italy, it was a 30 percentage point spread (roughly).

I found the data in the Supplementary Materials yesterday (July 13, 2021). When I looked this morning, that information was no longer there but you will find what appears to be the questionnaire. I wonder if this removal is temporary or permanent and, if permanent, I wonder why it was removed.

Participants for the Canadian portion of the survey were supplied by Dynata, a US-based market research company. Here’s the company’s Wikipedia entry and its website.

Information about how participants were recruited was also missing this morning (July 14, 2021).

Genome British Columbia (Genome BC)

I was a little surprised when I couldn’t find any information about the program or the project on the Genome BC website as the organization is listed as a funder.

There is a ‘Genomics and Society’ tab (seems promising, eh?) on the homepage where you can find the answer to this question: What is GE³LS Research?,

GE3LS research is interdisciplinary, conducted by researchers across many disciplines within social science and humanities, including economics, environment, law, business, communications, and public policy.

There’s also a GE3LS Research in BC page titled Project Search; I had no luck there either.

It all seems a bit mysterious to me and, just in case anything else disappears off the web, here’s a July 13, 2021 news item about the research on phys.org as backup to what I have here.

Precision targeting of the liver for gene editing

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Apparently the magic is in the lipid nanoparticles. A March 1, 2021 news item on Nanowerk announced research into lipid nanoparticles as a means to deliver CRISPR (clustered regularly interspaced short palindromic repeats) to specific organs (Note: A link has been removed),

The genome editing technology CRISPR has emerged as a powerful new tool that can change the way we treat disease. The challenge when altering the genetics of our cells, however, is how to do it safely, effectively, and specifically targeted to the gene, tissue and organ that needs treatment.

Scientists at Tufts University and the Broad Institute of Harvard [University] and MIT [Massachusetts Institute of Technology] have developed unique nanoparticles comprised of lipids — fat molecules — that can package and deliver gene editing machinery specifically to the liver.

In a study published in the Proceedings of the National Academy of Sciences [PNAS] (“Lipid nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-specific in vivo genome editing of Angptl3”), they have shown that they can use the lipid nanoparticles (LNPs) to efficiently deliver the CRISPR machinery into the liver of mice, resulting in specific genome editing and the reduction of blood cholesterol levels by as much as 57% — a reduction that can last for at least several months with just one shot.

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A March 2, 2021 Tufts University news release (also on EurekAlert but published March 1, 2021), which originated the news item, provides greater insight into and technical detail about the research,

The problem of high cholesterol plagues more than 29 million Americans, according to the Centers for Disease Control and Prevention. The condition is complex and can originate from multiple genes as well as nutritional and lifestyle choices, so it is not easy to treat. The Tufts and Broad researchers, however, have modified one gene that could provide a protective effect against elevated cholesterol if it can be shut down by gene editing.

The gene that the researchers focused on codes for the angiopoietin-like 3 enzyme (Angptl3). That enzyme tamps down the activity of other enzymes – lipases – that help break down cholesterol. If researchers can knock out the Angptl3 gene, they can let the lipases do their work and reduce levels of cholesterol in the blood. It turns out that some lucky people have a natural mutation in their Angptl3 gene, leading to consistently low levels of triglycerides and low-density lipoprotein (LDL) cholesterol, commonly called “bad” cholesterol, in their bloodstream without any known clinical downsides.

“If we can replicate that condition by knocking out the angptl3 gene in others, we have a good chance of having a safe and long term solution to high cholesterol,” said Qiaobing Xu, associate professor of biomedical engineering at Tufts’ School of Engineering and corresponding author of the study. “We just have to make sure we deliver the gene editing package specifically to the liver so as not to create unwanted side effects.”

Xu’s team was able to do precisely that in mouse models. After a single injection of lipid nanoparticles packed with mRNA coding for CRISPR-Cas9 and a single-guide RNA targeting Angptl3, they observed a profound reduction in LDL cholesterol by as much as 57% and triglyceride levels by about 29 %, both of which remained at those lowered levels for at least 100 days. The researchers speculate that the effect may last much longer than that, perhaps limited only by the slow turnover of cells in the liver, which can occur over a period of about a year. The reduction of cholesterol and triglycerides is dose dependent, so their levels could be adjusted by injecting fewer or more LNPs in the single shot, the researchers said.

By comparison, an existing, FDA [US Food and Drug Administration]-approved version of CRISPR mRNA-loaded LNPs could only reduce LDL cholesterol by at most 15.7% and triglycerides by 16.3% when it was tested in mice, according to the researchers.

The trick to making a better LNP was in customizing the components – the molecules that come together to form bubbles around the mRNA. The LNPs are made up of long chain lipids that have a charged or polar head that is attracted to water, a carbon chain tail that points toward the middle of the bubble containing the payload, and a chemical linker between them. Also present are polyethylene glycol, and yes, even some cholesterol – which has a normal role in lipid membranes to make them less leaky – to hold their contents better.

The researchers found that the nature and relative ratio of these components appeared to have profound effects on the delivery of mRNA into the liver, so they tested LNPs with many combinations of heads, tails, linkers and ratios among all components for their ability to target liver cells. Because the in vitro potency of an LNP formulation rarely reflects its in vivo performance, they directly evaluated the delivery specificity and efficacy in mice that have a reporter gene in their cells that lights up red when genome editing occurs. Ultimately, they found a CRISPR mRNA-loaded LNP that lit up just the liver in mice, showing that it could specifically and efficiently deliver gene-editing tools into the liver to do their work.

The LNPs were built upon earlier work at Tufts, where Xu and his team developed LNPs with as much as 90% efficiency in delivering mRNA into cells. A unique feature of those nanoparticles was the presence of disulfide bonds between the long lipid chains. Outside the cells, the LNPs form a stable spherical structure that locks in their contents. When they are inside a cell, the environment within breaks the disulfide bonds to disassemble the nanoparticles. The contents are then quickly and efficiently released into the cell. By preventing loss outside the cell, the LNPs can have a much higher yield in delivering their contents.

“CRISPR is one of the most powerful therapeutic tools for the treatment of diseases with a genetic etiology. We have recently seen the first human clinical trail for CRISPR therapy enabled by LNP delivery to be administered systemically to edit genes inside the human body. Our LNP platform developed here holds great potential for clinical translation,” said Min Qiu, post-doctoral researcher in Xu’s lab at Tufts.  “We envision that with this LNP platform in hand, we could now make CRISPR a practical and safe approach to treat a broad spectrum of liver diseases or disorders,” said Zachary Glass, graduate student in the Xu lab. Qiu and Glass are co-first authors of the study.

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

Lipid nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-specific in vivo genome editing of Angptl3 by Min Qiu, Zachary Glass, Jinjin Chen, Mary Haas, Xin Jin, Xuewei Zhao, Xuehui Rui, Zhongfeng Ye, Yamin Li, Feng Zhang, and Qiaobing Xu. PNAS March 9, 2021 118 (10) e2020401118 DOI: https://doi.org/10.1073/pnas.2020401118

This paper appears to be behind a paywall.

Toronto’s ArtSci Salon and The Mutant Project March 15, 2021

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The Mutant Project is both a book (The Mutant Project: Inside the Global Race to Genetically Modify Humans) and an event about gene editing with special reference to the CRISPR (clustered regularly interspaced short palindromic repeats) twins, Lulu and Nana. The event is being held by Toronto’s ArtSci Salon. Here’s more from their March 3, 2021 announcement (received via email),

The Mutant Project

A talk and discussion with Eben Kirksey

Discussants:

Dr. Elizabeth Koester, Postdoctoral fellow, Department of History, UofT [University of Toronto]

Vincent Auffrey, PhD student, IHPST [Institute for the History and Philosophy of Science and Technology], UofT

Fan Zhang, PhD student, IHPST, UofT

This event will be streamed on Zoom and on Youtube

Here is the link to register to Zoom on the 15th:

https://utoronto.zoom.us/meeting/registe/tZErcemoqzwrG9foNF5Ud86uJXdNeIzQSfDw

Event on FB: https://www.facebook.com/events/4033393163381012/

YouTube: https://www.youtube.com/watch?v=pEFfj3Ovsfk&feature=youtu.be

At a conference in Hong Kong in November 2018, Dr. He Jiankui announced that he had created the first genetically modified babies—twin girls named Lulu and Nana—sending shockwaves around the world. A year later, a Chinese court sentenced Dr. He to three years in prison for “illegal medical practice.”

As scientists elsewhere start to catch up with China’s vast genetic research program, gene editing is fueling an innovation economy that threatens to widen racial and economic inequality. Fundamental questions about science, health, and social justice are at stake: Who gets access to gene editing technologies? As countries loosen regulations around the globe, from the U.S. to Indonesia, can we shape research agendas to promote an ethical and fair society?

Join us to welcome Dr. Kirksey, who will discuss key topics from his book “The Mutant Project”.

The talk will be followed by a Q&A

EBEN KIRKSEY is an American anthropologist who finished his latest book as a Member of the Institute for Advanced Study in Princeton, New Jersey. He has been published in Wired, The Atlantic, The Guardian and The Sunday Times. He is sought out as an expert on science in society by the Associated Press, The Wall Street Journal, The New York Times, Democracy Now, Time and the BBC, among other media outlets. He speaks widely at the world’s leading academic institutions including Oxford, Yale, Columbia, UCLA, and the International Summit of Human Genome Editing, plus music festivals, art exhibits, and community events. Professor Kirksey holds a long-term position at Deakin University in Melbourne, Australia. For more information, please visit https://eben-kirksey.space/.

Elizabeth Koester currently holds a SSHRC [Social Science and Humanities Research Council of Canada] Postdoctoral Fellowship in the Department of History at the University of Toronto. After practising law for many years, she undertook graduate studies in the history of medicine at the Institute for the History and Philosophy of
Science and Technology at the University of Toronto and was awarded a PhD in 2018. A book based on her dissertation, In the Public Good: Eugenics and Law in Ontario, will be published by McGill-Queen’s University Press and is anticipated for Fall 2021.

Vincent Auffrey is pursuing his PhD at the Institute for the History of Philosophy of Science and Technology (IHPST) at the University of Toronto. His focus is set primarily on the social history of medicine and the history of eugenics in Canada. Secondary interests include the histories of scientific racism and of anatomy, and the interplay between knowledge and power.

Fan Zhang is a PhD student at the History of Philosophy of Science and Technology (IHPST) at the University of Toronto

Kirksey’s eponymous website,

CRISPR technology is like a pair of scissors and a dimmer switch?

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The ‘pair of scissors’ analogy is probably the most well known of the attempts to describe how the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing system works. It seems a new analogy is about to be added according to a January 19 2021 news item on ScienceDaily (Note: This October 30, 2019 posting features more CRISPR analogies),

In a series of experiments with laboratory-cultured bacteria, Johns Hopkins scientists have found evidence that there is a second role for the widely used gene-cutting system CRISPR-Cas9 — as a genetic dimmer switch for CRISPR-Cas9 genes. Its role of dialing down or dimming CRISPR-Cas9 activity may help scientists develop new ways to genetically engineer cells for research purposes.

Here’s an image illustrating the long form of the tracrRNA or ‘dimmer switch’ alongside the more commonly used short form,

Caption: Left – a schematic of the long form of the tracrRNA used by the CRISPR-Cas9 system in bacteria; Right – the standard guide RNA used by many scientists as part of the gene-cutting CRISPR-Cas9 system. Credit: Joshua Modell, Rachael Workman and Johns Hopkins Medicine

A January 19 ,2021 Johns Hopkins Medicine news release (also on EurekAlert), which originated the news item, explains about CRISPR and what the acronym stands for, as well as, giving more details about the discovery,

First identified in the genome of gut bacteria in 1987, CRISPR-Cas9 is a naturally occurring but unusual group of genes with a potential for cutting DNA sequences in other types of cells that was realized 25 years later. Its value in genetic engineering — programmable gene alteration in living cells, including human cells — was rapidly appreciated, and its widespread use as a genome “editor” in thousands of laboratories worldwide was recognized in the awarding of the Nobel Prize in Chemistry last year to its American and French co-developers.

CRISPR stands for clustered, regularly interspaced short palindromic repeats. Cas9, which refers to CRISPR-associated protein 9, is the name of the enzyme that makes the DNA slice. Bacteria naturally use CRISPR-Cas9 to cut viral or other potentially harmful DNA and disable the threat, says Joshua Modell, Ph.D., assistant professor of molecular biology and genetics at the Johns Hopkins University School of Medicine. In this role, Modell says, “CRISPR is not only an immune system, it’s an adaptive immune system — one that can remember threats it has previously encountered by holding onto a short piece of their DNA, which is akin to a mug shot.” These mug shots are then copied into “guide RNAs” that tell Cas9 what to cut.

Scientists have long worked to unravel the precise steps of CRISPR-Cas9’s mechanism and how its activity in bacteria is dialed up or down. Looking for genes that ignite or inhibit the CRISPR-Cas9 gene-cutting system for the common, strep-throat causing bacterium Streptococcus pyogenes, the Johns Hopkins scientists found a clue regarding how that aspect of the system works.

Specifically, the scientists found a gene in the CRISPR-Cas9 system that, when deactivated, led to a dramatic increase in the activity of the system in bacteria. The product of this gene appeared to re-program Cas9 to act as a brake, rather than as a “scissor,” to dial down the CRISPR system.

“From an immunity perspective, bacteria need to ramp up CRISPR-Cas9 activity to identify and rid the cell of threats, but they also need to dial it down to avoid autoimmunity — when the immune system mistakenly attacks components of the bacteria themselves,” says graduate student Rachael Workman, a bacteriologist working in Modell’s laboratory.

To further nail down the particulars of the “brake,” the team’s next step was to better understand the product of the deactivated gene (tracrRNA). RNA is a genetic cousin to DNA and is vital to carrying out DNA “instructions” for making proteins. TracrRNAs belong to a unique family of RNAs that do not make proteins. Instead, they act as a kind of scaffold that allows the Cas9 enzyme to carry the guide RNA that contains the mug shot and cut matching DNA sequences in invading viruses.

TracrRNA comes in two sizes: long and short. Most of the modern gene-cutting CRISPR-Cas9 tools use the short form. However, the research team found that the deactivated gene product was the long form of tracrRNA, the function of which has been entirely unknown.

The long and short forms of tracrRNA are similar in structure and have in common the ability to bind to Cas9. The short form tracrRNA also binds to the guide RNA. However, the long form tracrRNA doesn’t need to bind to the guide RNA, because it contains a segment that mimics the guide RNA. “Essentially, long form tracrRNAs have combined the function of the short form tracrRNA and guide RNA,” says Modell.

In addition, the researchers found that while guide RNAs normally seek out viral DNA sequences, long form tracrRNAs target the CRISPR-Cas9 system itself. The long form tracrRNA tends to sit on DNA, rather than cut it. When this happens in a particular area of a gene, it prevents that gene from expressing, — or becoming functional.

To confirm this, the researchers used genetic engineering to alter the length of a certain region in long form tracrRNA to make the tracrRNA appear more like a guide RNA. They found that with the altered long form tracrRNA, Cas9 once again behaved more like a scissor.

Other experiments showed that in lab-grown bacteria with a plentiful amount of long form tracrRNA, levels of all CRISPR-related genes were very low. When the long form tracrRNA was removed from bacteria, however, expression of CRISPR-Cas9 genes increased a hundredfold.

Bacterial cells lacking the long form tracrRNA were cultured in the laboratory for three days and compared with similarly cultured cells containing the long form tracrRNA. By the end of the experiment, bacteria without the long form tracrRNA had completely died off, suggesting that long form tracrRNA normally protects cells from the sickness and death that happen when CRISPR-Cas9 activity is very high.

“We started to get the idea that the long form was repressing but not eliminating its own CRISPR-related activity,” says Workman.

To see if the long form tracrRNA could be re-programmed to repress other bacterial genes, the research team altered the long form tracrRNA’s spacer region to let it sit on a gene that produces green fluorescence. Bacteria with this mutated version of long form tracrRNA glowed less green than bacteria containing the normal long form tracrRNA, suggesting that the long form tracrRNA can be genetically engineered to dial down other bacterial genes.

Another research team, from Emory University, found that in the parasitic bacteria Francisella novicida, Cas9 behaves as a dimmer switch for a gene outside the CRISPR-Cas9 region. The CRISPR-Cas9 system in the Johns Hopkins study is more widely used by scientists as a gene-cutting tool, and the Johns Hopkins team’s findings provide evidence that the dimmer action controls the CRISPR-Cas9 system in addition to other genes.

The researchers also found the genetic components of long form tracrRNA in about 40% of the Streptococcus group of bacteria. Further study of bacterial strains that don’t have the long form tracrRNA, says Workman, will potentially reveal whether their CRISPR-Cas9 systems are intact, and other ways that bacteria may dial back the CRISPR-Cas9 system.

The dimmer capability that the experiments uncovered, says Modell, offers opportunities to design new or better CRISPR-Cas9 tools aimed at regulating gene activity for research purposes. “In a gene editing scenario, a researcher may want to cut a specific gene, in addition to using the long form tracrRNA to inhibit gene activity,” he says.

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

A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression by Rachael E. Workman, Teja Pammi, Binh T.K. Nguyen, Leonardo W. Graeff, Erika Smith, Suzanne M. Sebald, Marie J. Stoltzfus, Chad W. Euler, Joshua W. Modell. Cell DOI:https://doi.org/10.1016/j.cell.2020.12.017 Published Online:J anuary 08, 2021

This paper is behind a paywall.