The CRISPR wrecking ball revealed

April 2019

US government information on genome (gene) editing describes it as a "group of technologies used by scientists to change an organism's DNA".

The most popular member of this group is 'Cas9', an enzyme which cuts DNA and can be designed to home in on a precise location in the genome [1,2]. Recently, a variant of this enzyme, 'Cas12a', has been developed: this seems to cut in a way that causes less disturbance at the cut ends of the DNA.

With regard to gene-edited crops, a team of Chinese scientists took a belated, close look at all the DNA changes arising in a novel rice model and what part of the technology caused them.

During natural reproduction, 'wild type' rice plants acquire around 41 small 'spontaneous mutations' (altered, added or deleted bits of the DNA nucleic acid chain) in their DNA. Since healthy plants are well able to correct any such changes which don't suit them, these mutations would seem to be part of the natural variation and flexibility important to achieving stability in the newly bred organism.

Over and above this apparently natural background of small DNA mutations during breeding, the gene editing process includes a number of potentially DNA-damaging procedures.

The first stage of gene-editing technology is to take a bit of a wild-type plant (such as a seed) and pull it to bits to extract some cells. These cells are persuaded to grow in an artificial medium containing nutrients and growth promoters. At this stage, the rice is no longer a plant or an organism. Forcing unnatural cell growth in this way is destabilising, and the cells which survive the process can acquire up to 231 small DNA mutations.

These cells in culture are then infected with a GM bacterial plant pathogen, Agrobacterium. Wild-type Agrobacterium naturally introduces its own DNA into the plant genome for the purpose of creating a gall (cancer) of plant tissue in which the bacterium can live. The GM Agrobacterium, has had its gall-inducing DNA removed and replaced with gene-editing DNA which therefore becomes inserted into the plant instead. This disease-causing stage of gene-editing technology results in up to 41 more small DNA mutations (this could be a low estimate, see [3])

Growth conditions of the infected cell culture are next altered to induce plantlet formation. Some of the plantlets will be gene-edited as required, some will be failures due to non-editing, non-expression of the edit, or due to the presence of mixed (GM/non-GM) cell types; all of these (number unknown) failures are binned.

The successful gene-edited plantlets are developed into plants (they become organisms again, albeit artificial ones).

Further small DNA mutations, due solely to the gene-edit, were recorded in one of the twelve plantlets altered with Cas9 and none of the three plantlets altered with Cas12a.

Besides the desired edit, the progeny of the experimental plantlets retained the Agrobacterium-mediated Cas9/Cas12a constructs.

Comment. There was no evidence at this (1st generation) stage that the Cas editors carried on causing mutations elsewhere in the genome. However, what could evolve once the plants are in the field under environmental stress and pathogen attack is not predictable.

The Chinese team commented that the precision of the genome edit by Cas enzymes is "stunning" in contrast to conventional breeding, and concluded that this proven precision supports the industry-friendly regulation of the Cas-edit itself (as in America), not the editing process (as in the EU).

OUR COMMENT

This conclusion can only be described as 'stunning': "From an omnigenic viewpoint, a tiny gene 'edit' can be just as unpredictable and unbalancing in its effects as a wholesale DNA insertion" [3]. Given the wrecked, multiply-mutated, infected, non-organismic origin in which that 'precise' Cas edit (plus the Cas construct) resides, it looks more than ever essential to regulate the process.

Moreover, the DNA mutations (large or small) as measured in this study are a very small part of the potential functional disruption which GM will inflict on the genome. Epigenetic mutations* and their consequences can also be a minefield of harm to the GM crop, to the environment and to the consumer.

The biotech industry PR machine, intent on proving how "precise" it's technologies are, is busy with "shouty" WORDS emphasising the absurdly back-to-front conclusion in what is otherwise an excellent and important piece of scientific research. Keep your regulator's eye on the gene-editing ball: the GM process is disruptive at every stage and must be regulated.

Background

[1] CRISPR/Cas9 GENE EDITING - March 2016

[2] CRISPR HAS IN-BUILT IMPRECISION - April 2018

[3] LET'S THINK OMNIGENICS - April 2019

*The epigenome consists of the DNA-associated biochemical and structural aspects of the genome which alter DNA function; these are not permanent but are heritable.

SOURCES:

• What are genome editing and CRISPR-Cas9? https://ghr.nlm.nih.gov/primer/genomicresearch/genomeediting
• Xu Tang, et al., 2018, A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 (Cas12a) nucleases in rice, Genome Biology 19:84
• New study claimed to show safety of CRISPR shows the opposite, GM Watch, 29.01.19
• Bekir Ulker, Worry about tissue culture as a source of mutations and not the genome editing itself! www.linkedin.com
• www.agrojector.com

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