by Jonathan Latham, PhD

The standard gene-editing tool, CRISPR-Cas9, frequently produces a type of DNA mutation that ordinary genetic analysis misses, claims new research published in the journal Science Advances. In describing these findings the researchers called such oversights “serious pitfalls” of gene editing (Skryabin et al., 2020). In all, the new results suggest that gene-editing is more error-prone than thought and, further, that identifying and discarding defective and unwanted outcomes is not as easy as generally supposed.

Derived originally from the bacterium streptococcus pyogenes, CRISPR-Cas9 is a DNA cutting and targeting system. CRISPR stands for c lustered r egularly i nterspaced s hort p alindromic r epeats and refers to the RNA molecule that is the targeting component of the system. This CRISPR RNA is sometimes also referred to as the guide RNA. The Cas9 component is a nuclease, that is, an enzyme that cuts DNA. Thus, in the editing process, the Cas9 enzyme is guided to the intended cut site by the CRISPR RNA. The whole assembly is often just called CRISPR.

Other gene-editing methods exist (e.g. Zn Finger, TALENs). However, because of the flexibility of its RNA targeting mechanism, CRISPR in particular has been the subject of enormous excitement in the biotech and agricultural research sectors.

CRISPR has mostly been used to create genetic mutations or to insert foreign DNA at desired locations in a genome. Nevertheless, other applications, like gene drives, have also been mooted. Despite the excitement, as Friends of the Earth has summarised, just a tiny handful of commercial gene-edited products can be found on the market.

For many uses, however, gene-editing with CRISPR is insufficiently precise and a great deal of research is currently oriented towards fixing this defect.

Much of CRISPR’s lack of precision derives from the fact that, though it is called ‘editing’, CRISPR and related techniques are cutting enzymes only. They have no DNA repair function. This means that when repairs are made to the DNA at the cut site (and the cut must be repaired for the cell to survive) they are largely out of the control of the experimenter. Ten independent editing events will therefore give ten different mutations at the same location in the genome.

Thus, at a very basic level, each mutation created at the target site is likely to be unique. Even to the extent, as we reported, that DNA from other species may end up being unexpectedly incorporated into the edited genome.

To add to this uncertainty, different genome locations, different cell types, different species, and different versions of CRISPR, can all influence the kinds of genetic alteration found at the target site.

In some applications–primarily basic research–lack of precision of this kind is not necessarily a major problem. In crop breeding, for example, cells or organisms containing undesirable alterations or off-target mutations can, in theory, be detected and discarded.

But in many applications, primarily in medicine and commercial products, only more-or-less-complete precision is acceptable, for reasons of safety. Inaccurate editing of human cells in an early gene therapy trial once resulted in 2 of 11 treated children developing leukaemia due to off-target effects and led to the trial being shut down.

The question of whether researchers and/or developers of edited organisms could or would adequately detect and discard undesirable mutations is a live concern. Recombinetics is a commercial company that, in 2016, created a hornless cow it claimed was the intended result of a precise gene edit. But FDA researchers who examined the company’s own DNA sequence data were subsequently able to show that both of the independently edited calves contained, at the site of the edit, entire antibiotic resistance genes (Norris et al., 2020).

By the time FDA was able to show this, however, offspring of the calves where already incorporated into a Brazilian breeding program. This breeding program has now been abandoned.

The new research, published on Feb 12th, directly addresses whether CRISPR researchers can, in fact, detect aberrant edits.

The German and Chinese researchers edited mouse oocytes (i.e. embryos) with the added step (compared to simple cutting) of adding a stretch of DNA (the donor DNA) which they hoped would become integrated at the cut site.

What they unexpectedly found, however, is that, at a high proportion of target sites, complex insertions of the desired DNA occurred. Rather than simply integrating single copies of the donor DNA into the cut site, DNA integrations were commonly head-to-tail arrangements of multiple copies. As the paper states:

“Overall, we conclude that the repetitive head-to-tail integration of the donor DNA template is a common by-product of the CRISPR-Cas9-mediated HDR-based genome editing process, regardless of the donor DNA template size, sequence composition, or strandedness of the template (dsDNA or ssDNA).” [editor’s note: ds=double stranded; ss=single stranded]

By ‘common’ the researchers meant that, in one experiment, among 34 edited mice, six contained head-to-tail insertions. In other experiments 30 of 49 mice contained head-to-tail insertions.

In other words, complex and aberrant DNA insertions were common findings. Importantly, they occurred in multiple experiments, meaning this seems to be true regardless of what DNA was inserted or which stretch of the genome it was inserted into. This in itself is a very significant finding.

Even more notable, however, was that these complex genetic rearrangements were rarely detected by standard analytical methods. The authors called this finding “disturbing”.

They wrote:

“conventionally applied PCR analysis, in most cases, failed to identify these multiple integration events, which led to a high rate of falsely claimed precisely edited alleles.”

Undetected, such aberrant events “would undermine the validity of studies” according to the authors.

In experimental settings this is undoubtedly true. But for the general public a more important implication exists. With companies and biohackers hoping to bring genome-edited products rapidly (and without regulatory scrutiny) to the market, this research represents a significant cautionary tale; especially since the authors speculate that their results probably apply equally to other editing methods, such as TALENs and Zn Finger nucleases.

References

Boris V. Skryabin, Delf-Magnus Kummerfeld, Leonid Gubar, Birte Seeger, Helena Kaiser et al. (2020) Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9–mediated genome editing events. Science Advances 6 eaax2941 DOI: 10.1126/sciadv.aax2941

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