by Sayer Ji, Green Med Info:
The gene-editing of DNA inside living cells is considered by many to be the preeminent technological breakthrough of the new millennium. Researchers in medicine and agriculture have rapidly adopted it as a technique for discovering cell and organism functions. But its commercial prospects are much more complicated.
Gene-editing has many potential uses. These include altering cells to treat human disease, altering crops and livestock for breeding and agriculture. Furthermore, in a move that has been widely criticised, Chinese researcher He Jiankui claims to have edited human babies to resist HIV by altering a gene called CCR5.
For most commercial applications gene-editing’s appeal is simplicity and precision: it alters genomes at precise sites and without inserting foreign DNA. This why, in popular articles, gene-editing is often referred to as ‘tweaking’.
The tweaking narrative, however, is an assumption and not an established fact. And it recently suffered a large dent. In late July researchers from the US Food and Drug Administration (FDA) analysed the whole genomes of two calves originally born in 2016. The calves were edited by the biotech startup Recombinetics using a gene-editing method called TALENS (Norris et al., 2019). The two Recombinetics animals had become biotech celebrities for having a genetic change that removed their horns. Cattle without horns are known as ‘polled’. The calves are well-known because Recombinetics has insisted that its two edited animals were extremely precisely altered to possess only the polled trait.
However, what the FDA researchers found was not precision. Each of Recombinetics’ calves possessed two antibiotic resistance genes, along with other segments of superfluous bacterial DNA. Thus, apparently unbeknownst to Recombinetics, adjacent to its edited site were 4,000 base pairs of DNA that originated from the plasmid vector used to introduce the DNA required for the hornless trait.
The FDA finding has attracted some media attention; mainly focussed on the incompetence of Recombinetics. The startup failed to find (or perhaps look for) DNA it had itself added as part of the editing process. Following the FDA findings, Brazil terminated a breeding program begun with the Recombinetics animals.
An animal research facility
But FDA’s findings are potentially trivial besides another recent discovery about gene-editing: that foreign DNA from surprising sources can routinely find its way into the genome of edited animals. This genetic material is not DNA that was put there on purpose, but rather, is a contaminant of standard editing procedures.
These findings have not been reported in the scientific or popular media. But they are of great consequence from a biosafety perspective and therefore for the commercial and regulatory landscape of gene-editing. They imply, at the very least, the need for strong measures to prevent contamination by stray DNA, along with thorough scrutiny of gene-edited cells and gene-edited organisms. And, as the Recombinetics case suggests, these are needs that developers themselves may not meet.
Understanding sources of stray DNA
As far back as 2010 researchers working with human cells showed that a form of gene-editing called Zinc Finger Nuclease (ZFN) could result in the insertion of foreign DNA at the editing target site (Olsen et al., 2010). The origin of this foreign DNA, as with Recombinetics’ calves, was the plasmid vector used in the editing process.
Understanding the presence of plasmid vectors requires an appreciation of the basics of gene-editing, which, confusingly, are considerably distinct from what the word ‘editing’ means in ordinary English.
Ultimately, all DNA ‘editing’ is really the cutting of DNA by enzymes, called nucleases, that are supposed to act only at chosen sites in the genome of a living cell. This cut creates a double-stranded break that severs (and therefore severely damages) a chromosome. The enzymes most commonly used by researchers for this cutting are the Fok I enzyme (for TALENS type editing), Cas9 (for CRISPR), or Zinc Finger Nucleases (for ZFN).
Subsequent to this cutting event the cell effects a repair. In practice, this DNA repair is usually inaccurate because the natural repair mechanism in most cells is somewhat random. The result is called the ‘edit’. Researchers typically must select from many ‘edits’ to obtain the one they desire.
Like virtually all enzymes these nucleases are proteins. And like most proteins they are somewhat tricky to produce and relatively unstable once made. Typically, therefore, rather than produce the DNA cutting enzymes directly, researchers introduce vector plasmids into target cells. These vector plasmids are circular DNA molecules that code for the desired enzyme(s). (vector plasmid DNA may also code for the guide RNA that CRISPR editing techniques require). What this means, in practice, is that TALENS, Cas9 and the other cutting enzymes end up being produced by the target cell itself.
Introducing DNA rather than proteins is thus much easier, research-wise, but it has a downside: non-host (i.e. transgenic) DNA must be introduced into the cell that is to be edited and this DNA may end up in the genome.
Plasmid vectors are not simple. As well as specifying the nucleases, the vector plasmid used by Recombinetics contained antibiotic resistance genes, plus the lac Z gene, plus promoter and termination sequences for each of them, plus two bacterial origins of replication. Each of these DNA components comes from widely diverse microbes.
As Olsen et al. and the FDA showed, using both TALENS and ZFN types of DNA cutters can result in plasmid vector integration at the target site. In 2015 Japanese researchers showed that DNA edits made to mouse zygotes using the CRISPR method of gene editing are also vulnerable to unintended insertion of non-host DNA (Ono et al., 2015).
Since then, similar integrations of foreign DNA at the target site have been observed in many species: fruitflies (Drosophila melanogaster), medaka fish (Oryzias latipes), mice, yeast, Aspergillus (a fungus), the nematode C. elegans, Daphnia magna, and various plants (e.g. Jacobs et al., 2015; Li et al., 2015; Gutierrez-Triana et al., 2018).
Other sources of stray DNA
The vector plasmids themselves are not the only source of potential foreign DNA contamination in standard gene-editing methodologies.
Earlier this year the same Japanese group showed that DNA from the E. coli genome can integrate in the target organisms’ genome (Ono et al. 2019). Acquisition of E. coli DNA was found to be quite frequent. Insertion of long unintended DNA sequences occurred at 4% of the total number of edited sites and 21% of these were of DNA from the E. coli genome. The source of the E. coli DNA was traced back to the E. coli cells that were used to produce the vector plasmid. The vector plasmid, which is DNA, was contaminated with E. coli genome DNA. Importantly, the Japanese researchers were using standard methods of vector plasmid preparation.