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.
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.
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DNA 자르지 않고 유전자 교정하는 기술 개발
샘 스탠버그 미국 컬럼비아대 생화학및분자생물물리학과 교수(맨 오른쪽)가 이끄는 연구팀이 콜레라균에서 아이디어를 얻어 DNA를 자르지 않고도 원하는 부분에 유전자를 삽입할 수 있는 신기술을 개발했다. 컬럼비아대 제공
DNA를 자르지 않고도 원하는 유전자를 삽입할 수 있는 새로운 유전자가위 기술이 나왔다. ‘인테그레이트’라는 이름이 붙여진 이 기술은 샘 스탠버그 미국 컬럼비아대 생화학및분자생물물리학과 교수팀이 콜레라균이 숙주의 DNA에 유전자를 넣는 모습에서 영감을 얻어 개발했다. 연구결과는 국제학술지 ‘네이처’ 12일자(현지시간)에 발표됐다.
DNA에서 질환을 유발하는 유전자를 없애거나, 원하는 유전자를 넣기 위해 현재 과학자들이 가장 많이 사용하는 도구는 ‘크리스퍼 유전자 가위’다. 크리스퍼 유전자 가위는 DNA에서 유전자를 편집할 부분을 찾는 ‘가이드RNA’와 이 DNA를 잘라내는 ‘절단효소’로 이뤄져 있다.
유전자가위 기술 중 가장 최신인 3세대 유전자가위 ‘크리스퍼’ 기술은 가장 효율적이고 정확해 혁신적인 생명공학 기술로 주목받고 있다. 하지만 최근 DNA 손상 부위를 교정하는 과정에서 삽입한 유전자나 그 주변의 염기서열에 돌연변이가 일어날 수 있다는 연구결과가 나오고 있다.
연구팀은 콜레라를 일으키는 균인 비브리오 콜레라(Vibrio cholerae)가 숙주의 DNA에 자기 유전자를 삽입하는 과정(트랜스포존)에서 아이디어를 얻었다. 가이드RNA에 절단효소 대신 ‘유전자삽입효소(인테그레이즈)’를 붙이는 것이다. DNA에서 유전자를 삽입할 부분을 가이드RNA로 찾은 다음, DNA를 자르는 대신 효소를 이용해 원하는 유전자를 삽입하는 원리다.
연구팀은 대장균을 대상으로 실험한 결과 실제로 DNA를 자르지 않고도 원하는 부위에 염기 1만개 짜리 크기의 유전자를 넣는 데 성공했다.
연구를 이끈 스탠버그 교수는 “유전자 연구 실험이나 농작물에 원하는 유전자를 넣는 생명공학에서 활용 가능하다”며 “부작용을 거의 발생시키지 않을 것”이라고 말했다. 다만 새로 개발된 기술인만큼 기존 크리스퍼 유전자 가위보다 훨씬 효율적이고 안전한지 추가 연구를 통해 확인할 필요가 있다.
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