A scientific review hypes the potential of the gene-editing tool CRISPR but can't deny its problems, including unintended effects at the on-target site. Report: Claire Robinson
A review published earlier this year addresses the problematic tendency of CRISPR gene-editing tools to cause off-target mutations (damage to DNA) and asks whether this is a concern when it comes to developing gene-edited plants.
The authors of the review (see abstract below) bow to the near-universal CRISPR hype in concluding, "CRISPR/Cas is a revolutionary technology that enables modifying plant genomes with unprecedented precision". But they also admit to ongoing problems with the technique and state that "a lot more research is needed" to figure out the extent of its unintended effects in plants.
The key message of this review is that the main problem of CRISPR is its tendency to cause off-target DNA damage. The authors suggest that this problem can be solved by more precisely targeting the initial “edit” to the desired location in the organism’s DNA.
However, there is another problem with CRISPR that will never be solved by this approach – namely the unintended effects of on-target edits. These unintended effects result from the cell’s DNA repair process after the CRISPR has performed its “editing” function. Genetic engineers have no control over them.
The authors of the review, Florian Hahn and Vladimir Nekrasov, are based at Rothamsted Research in the UK. Rothamsted partners with GMO companies such as Syngenta and Bayer.
The review is oddly self-contradictory, claiming miraculous precision for CRISPR while at the same time acknowledging lack of precision and unintended effects.
As Hahn and Nekrasov state, CRISPR gene-editing tools "are not 100% precise" and are able to cut DNA at off-target sites that share similarity with the target sites.
However, they add that while CRISPR tools can produce many off-target mutations in animals, most of these tools are "highly precise when applied in plants".
Nevertheless, the authors admit, "a number of reports in plants clearly demonstrated that CRISPR/Cas is capable of introducing mutations into off-target sites, which share a significant similarity with targets". In addition, they cite a study that found that the tissue culture process, which is done with all genetically modified (including gene-edited) plants, is a major source of mutations.
They recommend using certain gene-editing tools that have been found to perform better than others, in that they more precisely target the DNA cut and, based on limited evidence, reduce the number of off-target effects.
But in the authors’ own words, these tools are only "expected to be more precise in plants". In other words, they are not yet proven to be precise across a range of plants. And as Hahn and Nekrasov admit, when one type of "high fidelity" CRISPR tool was applied in Arabidopsis plants, off-target effects were indeed reduced, but so was its efficiency in producing the desired edit. That's a problem for genetic engineers.
The authors discuss the use of CRISPR editing tools in the form of a type of protein complex called ribonucleoproteins (RNPs) as an option for reducing off-target effects in plants. RNPs have attracted interest among CRISPR researchers because they can be easily reprogrammed to target any desired location in the genome that’s intended to be edited. The review authors note that this has been applied in several plants. But unfortunately for those gene-editing enthusiasts who would prefer to leave behind the controversy around old-style transgenic GM, which introduces “foreign” DNA inserted by a bacterium called Agrobacterium, the RNP method just doesn’t work in a lot of plants. In Hahn and Nekrasov’s words, “Many plant species are only stably transformable via Agrobacterium-mediated transformation, which is incompatible with the RNP strategy”.
Hahn and Nekrasov suggest that the most effective strategy to avoid off-target effects when using CRISPR is to select the right target sites for cutting, based on knowledge of the genome sequence of the species. The best sites to select are the ones with a minimum number of off-targets, as predicted by computer algorithms. In addition, for each gene-editing transformation "event", single guide RNAs (sgRNA, the molecule that guides the editing tool to the site in the DNA that is to be cut or "edited") should be designed with the minimum number of predicted off-targets.
Off-target effects not the only issue
The review authors correctly state that off-target effects are not the only concern with CRISPR. Even if researchers eventually find a way of precisely targeting the editing cut to a specific site in the DNA while excluding other non-target sites, unintended changes can also happen at the site of the intended DNA cut. Citing a study in mouse and human cells, the authors write, "Recently, Kosicki et al. have reported on unintended on-target changes, such as large chromosomal deletions, insertions and inversions, in mouse embryonic stem (ES) cells and human differentiated cells."
Such chromosomal rearrangements are not always easy to detect and thus many published studies on CRISPR use in plants may have missed them, the authors note. Other unintended changes, such as insertions of genetic material into the correctly targeted cut site, have been reported in plants, according to studies cited by the review authors.
Not surprisingly, Hahn and Nekrasov do not have a solution to the problem of unintended effects of CRISPR at the on-target site. They only state, “A lot more research is required to figure out if unintended on-target changes, such as large chromosomal insertions, deletions or inversions, induced by CRISPR/Cas are a common phenomenon in plants.”
Precision of initial editing cut is irrelevant
Since the publication of Hahn and Nekrasov’s review, a paper has appeared on a pre-publication website that challenges their implication that improving the precision of the initial CRISPR editing cut will make gene-editing any safer, more predictable, or more controllable.
This study in human cells, by Rubina Tuladhar and colleagues, investigated the outcomes in cells when the CRISPR system was used to knock-out a gene function by disrupting its normal base unit sequence. This disruption takes the form of insertions and/or deletions of relatively small stretches of DNA base units ("indels"). Indels are produced by the DNA repair process known as non-homologous end joining (NHEJ). NHEJ is activated within the cell nucleus after the CRISPR has made its double-strand DNA break, with the aim of repairing the cut ends of the DNA molecule.
Tuladhar and colleagues showed that instead of the intended outcome of totally destroying the function of a CRISPR-targeted gene, in 50% of the cell lines investigated, the indels resulted in an alteration of the gene’s DNA base unit sequence, so that it now produced new types of mRNAs (messenger RNA molecules) and proteins.
Commenting, the London-based molecular geneticist Dr Michael Antoniou said, "These unintended effects at the desired edited site take place after the gene-editing tool has performed its function and result from the innate DNA repair mechanisms of the cell. Therefore no matter how precise the targeting of the initial editing modification, these after-effects cannot be controlled or predicted by the genetic engineer. Therefore the argument that problems can be solved by modifying the CRISPR system to be more precisely targeted, thereby avoiding off-target editing, is spurious."
Cleaning up mutations
Hahn and Nekrasov state that off-target mutations in gene-edited plants can be "relatively easily" removed by taking affected plants out of the breeding program or by backcrossing them with elite varieties of non-GM plants.
But that's only the case with mutations that are visible to the genetic engineers – for example, those that cause deformities or poor growth. Mutations that cause more subtle changes, such as the production of toxins or allergens, are not easy to spot and could easily go undetected.
As Yves Bertheau, director of research at the French National Institute for Agricultural Research (INRA), has pointed out, numerous changes intentionally or unintentionally induced by gene-editing techniques can be transmitted to offspring and appear in the final marketed seed variety. Also, the number of backcrosses performed with elite varieties for marketed products is generally below the six that are theoretically necessary to obtain the desired minimum 95% level of genomic “purification”.
Review authors' conclusions – and ours
Hahn and Nekrasov conclude:
* There is a significant body of evidence suggesting that CRISPR/Cas is a precise genome editing tool when applied in plants.
* The authors present a good deal of evidence that CRISPR isn't precise and that these editing tools can cause unintended effects both at off-target sites and at the sites targeted for editing ("on-target sites").
Hahn and Nekrasov conclude:
* In the majority of cases, off-targeting can be avoided by designing specific sgRNAs with the minimum number of predicted off-targets.
* Even if this works as hoped to ensure the precise targeting of the intended editing cut, with no loss of efficiency – and there's no certainty of that – that won't prevent unintended effects occurring as a result of the intended "edit".
Hahn and Nekrasov conclude:
* "Although a lot more research is required to figure out if unintended on-target changes, such as large chromosomal insertions, deletions or inversions, induced by CRISPR/Cas are a common phenomenon in plants, there is no doubt that CRISPR/Cas is a revolutionary technology that enables modifying plant genomes with unprecedented precision."
* The authors claim precision regarding the initial targeted cut, but undermine this claim by stating in the same sentence that CRISPR and the associated tissue culture process also produce unintended effects at the targeted site, the extent and consequences of which are unknown.
Hahn and Nekrasov conclude:
* "Although CRISPR/Cas has its limitations, particularly when it comes to efficiency in many experimental setups in plants, in the future, it will certainly become a technology of choice for crop improvement and breeding, provided the regulatory and IP [intellectual property]-related issues are overcome."
* Whether CRISPR becomes a "technology of choice" relies on the ability of genetic engineers to overcome the "limitations" referred to in this same sentence, as well as many other factors, including what sort of agriculture we want to support.
As is common with scientific papers on GMOs, the propaganda messages in Hahn and Nekrasov's review are qualified and undermined by the actual data – including data that are presented in the review. The old saying, "The devil is in the detail", applies in this case.
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CRISPR/Cas precision: do we need to worry about off-targeting in plants?
Florian Hahn, Vladimir Nekrasov
Plant Cell Reports
April 2019, Volume 38, Issue 4, pp 437–441
First Online: 13 November 2018
The CRISPR/Cas technology has recently become the tool of choice for targeted genome modification in plants and beyond. Although CRSIPR/Cas offers a rapid and facile way of introducing changes at genomic loci of interest, its application is associated with off-targeting, i.e. introduction of unintended mutations at off-target sites within the genome, which has been reported frequently in the mammalian field. Here we summarise the current knowledge on the precision of CRISPR/Cas in plant systems and provide a summary of state-of the-art strategies for avoiding off-target mutations, as well as unintended on-target changes, in plants. These include using natural (e.g. Cas12a) or engineered (e.g. SpCas9-HF) CRISPR/Cas nucleases characterised by higher precision, as compared to the commonly used wild type SpCas9. In addition, we discuss the usage of CRISPR/Cas nucleases in the form of ribonucleoproteins (RNPs) as an option for reducing off-targeting in plants. Finally, we conclude that the most important factor for reducing CRISPR/Cas off-targeting remains careful selection of target sequences, for which we provide an overview of available online software tools and experimental guidance.