Further Challenges

Pleiotropy is not the only challenge for translating genome-editing tools into the clinical setting.

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Off-target effects and DNA repair-associated mutations are also significant concerns. Cas9-induced double-strand DNA breaks increase the risk of potentially-harmful insertion or deletion (indel) mutations via nonhomologous end-joining by DNA repair enzymes.

The exact scope of the problem remains unclear, but researchers agree that to some degree, CRISPR-Cas9 (and other genome-editing platforms, like TALENs and ZFN) induces not only intended edits to target genes, but also random indel mutations, which are sources of potentially-significant toxicity.

“The key limitations of the current genome-editing methods are the low efficiency of homologous repair and the challenges of delivering the editing materials to cells and organisms. Improving the former requires manipulating the cellular pathways of repair,” said Dana Carroll, PhD, distinguished professor in the department of biochemistry at the University of Utah School of Medicine. Dr Carroll is also a member of the Huntsman Cancer Institute’s nuclear control of cell growth and differentiation program in Salt Lake City. “Enhancing delivery is a problem that doesn’t have an obvious solution, which may depend on novel chemistries.”

One approach to reducing the frequency of indels might be to hybridize nucleotide editing systems from different species.

In yeast, at least, 1 such hybrid platform appears to be associated with less toxicity than the widely-used CRISPR-Cas9 platform, as indicated by yeast cell growth.3

Another approach to solving CRISPR-Cas9’s shortcomings, of course, would be to systematically screen different species’ adaptive immune systems in nature to identify those that might better meet human needs.

So far, only 3 to 5 basic types of CRISPR-Cas systems have been identified.4 But there are several CRISPR-Cas subtypes, and researchers are using variations among them to begin to piece together scenarios to explain how exactly CRISPR-Cas systems evolved.

Evolutionary “arms races” between prokaryotes and viruses have likely resulted in myriad such systems in nature, according to Dr Ewald. “I’d think there would be an arms race on the CRISPR proteins. If the virus hasn’t changed, then with CRISPR, the cell can destroy the virus. That puts selection pressure on viruses to change their [DNA] sequences. That could help explain why viruses evolve sequence variation so quickly. But you should expect arms races going on at the level of CRISPR proteins as well, so I think that we will find more variation.”

Evolutionary arms races among hosts and parasites or infectious organisms are not rare in nature, so it stands to reason that a large reservoir of such tools might await discovery. An immune defense system reminiscent of CRISPR was even recently described in a virus species that has to contend with viral parasites of its own, for example.5 A giant mimivirus that infects amoeba possesses a virophage resistant element, which appears to have resulted from an arms race between that species and smaller, virus-parasitizing “virophage” viruses.

Serendipity: the Future of Gene-editing?

It is open to question, however, whether the next big genome-editing breakthrough will come from systemically screening natural CRISPR and CRISPR-like systems.

“It is certainly hard to predict whether new gene editing systems will emerge from very broad studies of natural CRISPR systems,” Dr Carroll said. “I’m sure additional variants will turn up, but perhaps nothing dramatically different from what we’ve seen already.”

It’s just as likely that novel genome-editing tools will be discovered in the course of unrelated research, he believes.

“From meganucleases to ZFNs to TALENs to CRISPR, all our tools have come from investigations into how the world works, but not directed toward finding ways to make targeted breaks in DNA,” Dr Carroll explained.

Across the sciences, critical discoveries come from “unexpected places,” Dr Carroll said. “So we need to keep investigating nature on very broad fronts.”

Disclosures: the author has no relationships to report.


  1. Mohanraju P, Makarova KS, Zetsche B, et al. Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems. Science. 2016;353(6299):aad5147. doi: 10.1126/science.aad5147
  2. Abegglen LM, Caulin AF, Chan A, et al. Potential mechanisms for cancer resistance in elephants and comparative cellular response to DNA damage in humans. JAMA. 2015;314(17):1950-1860. doi: 10.1001/jama.2015.13134
  3. Nishida K, Arazoe T, Yachie N, et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science. 2016 Aug 4. doi: 10.1126/science.aaf8729 [Epub ahead of print]
  4. Makarova KS, Wolf YI, Alkhnbashi OS, et al. An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol. 2015;13(11):722-36. doi: 10.1038/nrmicro3569
  5. Levasseur A, Bekliz M, Chabriere E, Pontarotti P, La Scola B, Raoult D. MIMIVIRE is a defence system in mimivirus that confers resistance to virophage. Nature. 2016;531:249-252. doi: 10.1038/nature17146