Implications for food safety of gene-edited crops have not been investigated. Report: Claire Robinson

Yet another study has shown that the new GM technique CRISPR resulted in unexpected effects. The findings could have implications for the food safety of gene-edited crops produced using this genetic engineering tool.

In the new study (see abstract below), the authors 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 DNA base unit insertions and deletions ("indels"). Indels are produced by the DNA repair process known as non-homologous end joining (NHEJ), which gets activated in order to repair the cut ends of the DNA molecule once the CRISPR has made its double-strand DNA break.

The authors found 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) or proteins.

Indel formation by NHEJ repair occurs after the CRISPR has completed its function and thus takes place independently of this gene editing tool. Thus no matter how precise the genetic engineer makes the initial CRISPR-mediated cut in terms of location, the undesirable outcomes reported here can still take place. Similar outcomes can be expected when the CRISPR (contrary to the intended manipulation) cuts the DNA at an off-target site within non-targeted genes.

The new study is currently published on the pre-publication website bioRxiv so it is not yet peer-reviewed.

The study was performed in human cell lines. However, there is every reason to believe that unexpected outcomes of the type described in this study will take place in CRISPR'd plants where the aim is a gene knock-out by NHEJ-indel formation, since these processes are very similar in both plants and animals.

Many papers have been published showing unintended effects from the CRISPR process and other gene-editing tools. While most such studies are in animals and human cell lines, genetic errors have also been documented in many plants – e.g. Zhu and colleagues, 2017[1]; Wolt and colleagues, 2016[2]. Wolt's paper has a table of off-target effects in plants.

The concern with the production of novel proteins in food crops and food products by the unexpected CRISPR-NHEJ-indel process described in the new study is that it can result in altered plant biochemistry, leading to unexpected toxicity and/or allergenicity.

Numerous animal feeding studies (summarised in the book GMO Myths and Truths) show unexpected toxic or allergenic effects with the first generation of GM crops. There is every possibility that the second generation of GM crops – products of gene editing – will present similar problems. However, the necessary studies (including long-term animal feeding studies) have not been performed with gene-edited crops.

Commenting on the new study, London-based molecular geneticist Dr Michael Antoniou said, “The discoveries described in this study add to the increasing number of ways in which gene editing can go wrong. Regulators need to fully take on board these and other findings of off- and on-target unexpected outcomes from gene editing and subject all products produced with these methods to a comprehensive health risk assessment before considering market approval.”

Notes

1. Zhu C, Bortesi L, Baysal C, Twyman RM, Fischer R, Capell T, Schillberg S and Christou P (2017). Characteristics of genome editing mutations in cereal crops. Trends in Plant Science 22:38–52.

2. Wolt JD, Wang K, Sashital D and Lawrence-Dill CJ (2016). Achieving plant CRISPR targeting that limits off-target effects. The Plant Genome 9: doi: 10.3835/plantgenome2016.05.0047

UPDATE Sept 2019: This study by Tuladhar et al has now been published in Nature Communications.
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CRISPR/Cas9-based mutagenesis frequently provokes on-target mRNA misregulation
Rubina Tuladhar, Yunku Yeu, John Tyler Piazza, Zhen Tan, Jean Rene Clemenceau, Xiaofeng Wu, Quinn Barrett, Jeremiah Herbert, David H. Mathews, James Kim, Tae Hyun Hwang, Lawrence Lum (2019). bioRxiv, 583138.
https://www.biorxiv.org/content/10.1101/583138v1.full

Abstract

The introduction of insertion-deletions (INDELs) by activation of the error-prone non-homologous end-joining (NHEJ) pathway underlies the mechanistic basis of CRISPR/Cas9-directed genome editing. The ability of CRISPR/Cas9 to achieve gene elimination (knockouts) is largely attributed to the emergence of a pre-mature termination codon (PTC) from a frameshift-inducing INDEL that elicits non-sense mediated decay (NMD) of the mutant mRNA. Yet, the impact on gene expression as a consequence of CRISPR/Cas9-introduced INDELs into RNA regulatory sequences has been largely left uninvestigated. By tracking DNA-mRNA-protein relationships in a collection of CRISPR/Cas9-edited cell lines that harbor frameshift-inducing INDELs in various targeted genes, we detected the production of foreign mRNAs or proteins in ∼50% of the cell lines. We demonstrate that these aberrant protein products are derived from the introduction of INDELs that promote internal ribosomal entry, convert pseudo-mRNAs into protein encoding molecules, or induce exon skipping by disruption of exon splicing enhancers (ESEs). Our results using CRISPR/Cas9-introduced INDELs reveal facets of an epigenetic genome buffering apparatus that likely evolved to mitigate the impact of such mutations introduced by pathogens and aberrant DNA damage repair, and that more recently pose challenges to manipulating gene expression outcomes using INDEL-based mutagenesis.