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Biomedical Applications » Gene Therapy

Title: CRISPR/Cas9 Found to Cause Extensive Genetic Mutations in Cells
Publication date: September 12, 2018
Posting date: September 12, 2018

 THIRD WORLD NETWORK BIOSAFETY INFORMATION SERVICE


Dear Friends and Colleagues

CRISPR/Cas9 Found to Cause Extensive Genetic Mutations in Cells

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. Scientists at the Wellcome Sanger Institute 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.

Published in the journal Nature Biotechnology, the study found that CRISPR/Cas9 gene editing can cause greater genetic damage in cells than was previously thought. The researchers found that many of the cells had large genetic rearrangements such as DNA deletions and insertions.

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. In addition, some of these changes were too far away from the target site to be seen with standard genotyping methods. The researchers stressed 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.

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Item 1

REPAIR OF DOUBLE-STRAND BREAKS INDUCED BY CRISPR–CAS9 LEADS TO LARGE DELETIONS AND COMPLEX REARRANGEMENTS

By Michael KosickiKärt Tombergand Allan Bradley
Nature BiotechnologyVolume 36, pages 765–771 (2018)
16 July 2018
https://www.nature.com/articles/nbt.4192

CRISPR–Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR–Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR–Cas9 editing may have pathogenic consequences.


Item 2

CRISPR CAUSES GREATER GENETIC DAMAGE THAN PREVIOUSLY THOUGHT

GM Watch
17 July 2018
https://www.gmwatch.org/en/news/latest-news/18350-crispr-causes-greater-genetic-damage-than-previously-thought

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. Potential consequences could include triggering cancer.

Reported on 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.

As usual we see far more honesty about the off-target effects of CRISPR from genetic engineers in the field of medical research than we see from the plant genetic engineers. However, the technique as used in plants is the same, as are the mechanisms of DNA repair. These off-target effects in food plants could have possible knock-on effects on food safety, including unexpected toxicity and allergenicity.

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."

Food safety implications 

Commenting on the findings, London-based molecular biologist Dr Michael Antoniou said, "The main point to bear in mind about CRISPR is that all events after the CRISPR has cut the DNA occur independently of the CRISPR and are due to the cell's innate DNA repair machinery. Thus it doesn't matter how much you tweak the CRISPR to make it more 'specific' – you'll be faced with the same spectrum of downstream problems that are inherent to the non-homologous end joining (NHEJ) DNA repair system.

"These findings also do not bode well for the other genome editing tools (TALENs, ZFNs), as presumably the same things will happen after they too have produced a double strand DNA break and the NHEJ repair pathway kicks in.

"In addition, these findings suggest that we need to re-visit/re-evaluate the outcomes from NHEJ DNA repair in general. Do the outcomes reported in this latest study with CRISPR-generated double strand breaks also occur when the DNA is cut by other means, e.g. radiation or mutagenic chemicals? Or do they occur only when double strand breaks are brought about by editing tools?"

Dr Antoniou pointed out that some of our food plants have been produced through the older methods of radiation- or chemical-induced mutagenesis and if similar genetic damage to that seen when using CRISPR also occurs with the older methods, this could have implications for the food safety of those plants.


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