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Hijack of CRISPR defences by selfish genes holds clinical promise

Parasitic genetic elements called transposons carry CRISPR machinery that is normally used against them by bacterial cells. This paradox has now been explained, with implications for gene-therapy research.
Fyodor D. Urnov is at the Innovative Genomics Institute, Berkeley, California 94704, USA.
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When Hamlet is mortally wounded by Laertes’ poisoned blade in a fencing match, he switches weapons and strikes back, so that Laertes is killed by his own sword. Writing in Nature, Klompe et al.1 describe an equally dramatic weapon switch in biological warfare. They report that a molecular machine called Cascade, which bacteria use to defend themselves against genetic invaders, can also be used against them by some of those invaders. To add to the drama, this tiny instrument of war might eventually find itself serving a peaceful purpose: the genetic engineering of human cells to treat disease.

The genomes of bacteria are under constant assault from ‘selfish’ DNA segments (such as genes from bacterium-infecting viruses and mobile genetic elements), which enhance their own propagation and transmission, rather than their host’s. One type of mobile element is called a transposon. Some transposons carry just five genes, the sole function of which is to spread the transposon among bacteria2. The protein products of these genes work together to insert the transposon DNA into a specific spot in a bacterium’s genome at which insertion does not harm the host. The transposon thus becomes a permanent ‘passenger’ in that bacterium. When the opportunity arises, it transfers itself into one of the small, circular pieces of DNA that bacteria pass between each other to transfer genetic material, and can thereby move to a new host2.

Bacteria are armed with several defence systems against such parasites. One is known as CRISPR3, and works in a similar way to a ‘wanted’ poster of a criminal. When foreign DNA enters a bacterial cell, CRISPR chops it up and places a few fragments into the bacterial genome. These fragments are not dust-gathering war trophies, but ‘memories’ of past invasions: the bacterium copies them into short snippets of RNA, and hands them over to dedicated CRISPR-associated nuclease enzymes, of which Cas9 is the best studied4,5. These nucleases carry the RNA snippets and compare them with incoming DNA; if there is a match, the invading DNA is destroyed.

In 2017, a strange fact was reported by Peters et al.6: some transposons also carry genes for Cascade, a type of CRISPR defence system. This made no sense. Why would a parasitic genetic element need defence machinery that targets itself? Not all features of living things are Darwinian adaptations, but the puzzling prevalence of Cascade in transposons from many bacteria implied that it had to be there for a reason.

However, Peters et al. noted two peculiarities of the Cascade–transposon systems. First, although the Cascade machinery still recognized a target DNA by comparing it with an RNA snippet carried on a Cas-type protein, this machinery could not cut the DNA, and so was like a gun loaded with blanks. Second, the transposon carried all the usual genes required to integrate its DNA into a bacterial genome, but lacked the gene that directs that integration to the usual ‘safe for the host’ destination — thus preventing the Cascade gun from aiming at a specific target. Peters et al. hypothesized that these two minuses make a plus: perhaps the transposon uses Cascade to recognize a new DNA target in a bacterium, and then routes the integration of transposon DNA to that site?

Klompe and co-workers now provide a wealth of experimental data that prove and expand this idea. They show that the transposon can use the RNA-guided component of its Cascade passenger to direct Cascade to a particular position in a genome. They also report that, after recognizing the target DNA, Cascade directly binds to a protein (TniQ) that guides the insertion of the transposon to the new location in the genome (Fig. 1). This insertion is impressively specific — in all 25 cases studied by the authors, the transposon was delivered precisely and exclusively to the targeted address in the bacterial genome. Klompe and colleagues’ findings illuminate how evolution in microbes can morph, shuffle and combine components to come up with radical new solutions to problems — in this case, resulting in an RNA-guided transposition of DNA.

Figure 1 | Two ways in which genes can be inserted into chromosomes. a, In conventional gene editing, a nuclease enzyme (such as Cas9, part of the CRISPR defence mechanism in bacteria) is directed to a position on a chromosome by a guide RNA. The nuclease produces a double-strand break, which is repaired using the host cell’s machinery. The repair process is guided by a DNA template in which a therapeutic gene is flanked by stretches of DNA that are identical to the chromosome, and incorporates the gene into the chromosome10. b, Klompe et al.1 report that DNA elements called transposons use CRISPR machinery called Cascade (formed from Cas6, Cas7 and Cas8 proteins) to insert themselves into genomes. Cascade is directed to a chromosome by a guide RNA, but then binds a transposase-associated protein, TniQ, which in turn recruits the transposon and integrates it into the chromosome. This RNA-directed mechanism for DNA transposition avoids the need for double-strand breaks or long flanking sequences, and thus might help to address some of the shortcomings of conventional gene editing.

The work will inspire researchers working on an entirely different scientific front: the genetic engineering of humans to treat disease. Therapeutic genes are conventionally installed in humans using viruses that either persist outside the cell’s genome (which means that they are rapidly diluted when the cell divides) or land semi-randomly within the genomic DNA (which raises potential safety concerns)7. One solution to this problem is the technique called genome editing8,9 — in which an engineered nuclease, such as Cas9, is targeted to cut DNA at a position of interest to produce a double-strand break (DSB), which is then repaired using a template that facilitates the insertion of a gene at that position10 (Fig. 1a).

Although DSB-driven gene addition is useful, it has limitations. First, it works relatively inefficiently in non-dividing cells, many of which are potential targets for gene therapy. Second, the gene to be inserted must be flanked by DNA segments that match the sequence in the region of the genome into which it is being inserted, which takes up valuable space in the therapeutic agent. And third, the generation of a DSB has an associated risk11, albeit a manageable one. Both Peters et al.6 and Klompe et al. suggest that the reported transposons provide, in principle, a solution to all those issues: the transposon integration process does not require a DSB at the target (Fig. 1b), or flanking DNA in the therapeutic agent, and should work in non-dividing cells. Hence, it could be an attractive approach for human gene editing in the clinic.

However, a long checklist must be completed before clinical applications can be considered seriously. This list includes: showing that the process works efficiently at target genome positions in disease-relevant human cells (rather than in bacteria); demonstrating that it can integrate DNA fragments large enough to be clinically useful; proving its specificity in the human genome, which is about 1,000 times larger than a bacterial one; and developing ways to deliver the full complement of proteins associated with the integration process to cells without triggering the human immune response. This is a formidable workload, but a key lesson of the past 30 years of research into gene therapy is that most challenges of this type are eventually solved7,11,12. Therefore, a CRISPR system used by transposons to propagate themselves might well find itself repurposed for genetic medicine.

Nature 571, 180-181 (2019)

doi: 10.1038/d41586-019-01824-0

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