Tag Archives: Maria Jasin

Off-target CRISPR-Cas9 gene-editing changes closer to home than originally believed according to three studies

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Heidi Ledford’s June 25, 2020 article (Note: Links have been removed) for Nature focuses on three studies (not yet peer-reviewed) that viewed together suggest CRISPR (clustered regularly interspaced short palindromic repeats) gene-editing is less like using a pair of scissors to cut out unwanted mutations and more like using a catalyst (a chemical agent which increases chemical reactions) and getting unanticipated and unwatned reactions. Except, it’s an unpredictable catalyst.

A suite of experiments that use the gene-editing tool CRISPR–Cas9 to modify human embryos have revealed how the process can make large, unwanted changes to the genome at or near the target site. [emphasis mine]

The studies were published this month on the preprint server bioRxiv, and have not yet been peer-reviewed1,2,3. But taken together, they give scientists a good look at what some say is an underappreciated risk of CRISPR–Cas9 editing. Previous experiments have revealed that the tool can make ‘off target’ gene mutations far from the target site, but the nearby changes identified in the latest studies can be missed by standard assessment methods.

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These safety concerns are likely to inform the ongoing debate over whether scientists should edit human embryos to prevent genetic diseases — a process that is controversial because it creates a permanent change to the genome that can be passed down for generations. “If human embryo editing for reproductive purposes or germline editing were space flight, the new data are the equivalent of having the rocket explode at the launch pad before take-off,” says Fyodor Urnov, who studies genome editing at the University of California, Berkeley, but was not involved in any of the latest research.

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These studies,if borne out, offer new concerns (from Ledford’s June 25, 2020 article),

The changes are the result of DNA-repair processes harnessed by genome-editing tools. CRISPR–Cas9 uses a small strand of RNA to direct the Cas9 enzyme to a site in the genome with a similar sequence. The enzyme then cuts both strands of DNA at that site, and the cell’s repair systems heal the gap.

The edits occur during that repair: most often, the cell seals up the cut using an error-prone mechanism that can insert or delete a small number of DNA letters. If researchers provide a DNA template, the cell might sometimes use that sequence to mend the cut, resulting in a true rewrite. But broken DNA can also cause shuffling or loss of a large region of the chromosome.

Previous work using CRISPR in mouse embryos and other kinds of human cell had already demonstrated that editing chromosomes can cause large, unwanted effects4,5. But it was important to demonstrate the work in human embryos as well, says Urnov, because different cell types might respond to genome editing differently.

Such rearrangements could be missed in many experiments, which typically look for other unwanted edits, such as single DNA-letter changes or small insertions or deletions of only a few letters. The latest studies, however, looked specifically for large deletions and chromosomal rearrangements near the target site. [emphasis mine] “This is something that all of us in the scientific community will, starting immediately, take more seriously than we already have,” says Urnov. “This is not a one-time fluke.”

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Ledford’s article offers some description and analysis of each of the three papers.Note: All of the research was done with nonviable embryos. For anyone who wants to read the papers for themselves here are links and citations for each of the three,

Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human embryos by Gregorio Alanis-Lobato, Jasmin Zohren, Afshan McCarthy, Norah M.E. Fogarty, Nada Kubikova, Emily Hardman, Maria Greco, Dagan Wells, James M.A. Turner, Kathy K. Niakan. bioRxiv DOI: https://doi.org/10.1101/2020.06.05.135913 Posted: June 5, 2020

Reading frame restoration at the EYS locus, and allele-specific chromosome removal after Cas9 cleavage in human embryos by Michael V. Zuccaro, Jia Xu, Carl Mitchell, Diego Marin, Raymond Zimmerman, Bhavini Rana, Everett Weinstein, Rebeca T. King, Morgan Smith, Stephen H. Tsang, Robin Goland, Maria Jasin, Rogerio Lobo, Nathan Treff, Dieter Egli. bioRxiv DOI: https://doi.org/10.1101/2020.06.17.149237 Posted June 18, 2020

FREQUENT GENE CONVERSION IN HUMAN EMBRYOS INDUCED BY DOUBLE STRAND BREAKS by Dan Liang, Nuria Marti Gutierrez, Tailai Chen, Yeonmi Lee, Sang-Wook Park, Hong Ma, Amy Koski, Riffat Ahmed, Hayley Darby, Ying Li, Crystal Van Dyken, Aleksei Mikhalchenko, Thanasup Gonmanee, Tomonari Hayama, Han Zhao, Keliang Wu, Jingye Zhang, Zhenzhen Hou, Jumi Park, Chong-Jai Kim, Jianhui Gong, Yilin Yuan, Ying Gu, Yue Shen, Susan B. Olson, Hui Yang, David Battaglia, Thomas O’Leary, Sacha A. Krieg, David M. Lee, Diana H. Wu, P. Barton Duell, Sanjiv Kaul, Jin-Soo Kim, Stephen B. Heitner, Eunju Kang, Zi-Jiang Chen, Paula Amato, Shoukhrat Mitalipov. bioRxiv DOI: https://doi.org/10.1101/2020.06.19.162214 Posted June 20, 2020

These papers are open access.

A July 17, 2018 posting is probably the first time I featured work showing that CRISPR gene-editing can result in off-target effects; it was followed up by a September 20, 2019 posting on the topic.

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

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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),

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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.

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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.