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The domestication of Cas9 The enzyme Cas9 is used in genome editing to cut selected DNA sequences, but it also creates breaks at off-target sites. Protein engineering has now been used to make Cas9 enzymes that have minimal off-target effects. See Article p.490 In the present studies, Kleinstiver et al.1 and Slaymaker et al.2 set out or the past 30,000 years, to tame Cas9 using a thoughtful and humans have been genetically well-executed approach that relied Slaymaker et al. engineering the wolf through Complementary on an atom-by-atom understanding strand selective breeding, preserving some of how the enzyme binds to and cuts forms of genes and eliminating DNA8. (A similar study of how zinc others to produce the dog. Now, fingers bind DNA9 ultimately proKleinstiver et al. Target two studies (one on page 490 of this vided the basis for the first genomestrand issue1 and one in Science2) have used editing experiments.) The groups genetic engineering to tame a differreasoned that, by engineering Cas9 gRNA ent type of wild creature — a nuclease such that its interactions with the enzyme called Cas9. In doing so, they DNA backbone were weakened, Cas9 have markedly reduced the enzyme’s they could force the enzyme to rely undesirable natural tendencies, but to a greater extent on the gRNA– have preserved its ability to cut DNA Figure 1 | Taming a wild enzyme.  The Cas9 enzyme cuts specific DNA pairing to recognize and cut its in an RNA-guided manner. This feat DNA sequences, which it identifies using a guide RNA (gRNA) that target (Fig. 1). sequence in an unwound DNA double helix. of molecular domestication is great pairs with the chosen Kleinstiver and colleagues tested 1 news for practitioners of genome Kleinstiver et al. engineered Cas9 such that interactions between the the editing specificity of their resultediting, in which DNA sequences enzyme and the2backbone of the gRNA-paired DNA were weakened. ing enzyme, which they dubbed Slaymaker et al. engineered the contacts that the enzyme makes with in cells or organisms are changed to the complementary DNA single strand, which is not recognized by the high-fidelity Cas9 (Cas9-HF), in a scientists’ specifications efficiently gRNA. These modifications forced the engineered enzymes to rely to a cancer-cell line. They programmed and accurately; such precise editing greater extent on the gRNA for sequence recognition, thus improving Cas9-HF with gRNAs for seven difrequires highly targetable nucleases3. their binding specificity. (Adapted from ref. 1.) ferent stretches of human DNA: in In its natural ‘wild’ state, Cas9 is six cases it edited only the intended part of the bacterial immune systarget, and in the remaining stretch tem. When a bacterium is infected by a para- scale and scope of genome-editing applications the enzyme was weakly distracted by only one site such as a virus, the organism’s cellular for research purposes6, because the gRNA other position in the DNA. By comparison, machinery cuts up and retains pieces of the enables easy and relatively efficient enzyme wild-type Cas9 cut at multiple unintended invader’s DNA, storing the sequences in a programming. sequences when tested with gRNAs for these region of the bacterium’s own genome called Cas9 evolved to defend a bacterium that seven gene targets. Crucially, for 75% of targets a CRISPR locus4. Cas9 then polices the bacte- has a genome 1,800 times smaller than the tested, Cas9-HF was just as potent a genome rium for repeat invaders by carrying with it an human genome, and cutting DNA sequences editor as its wild ancestor. Slaymaker and RNA copy of a sequence stored in the CRISPR that are imperfectly matched to the gRNA is an colleagues followed the same overall principle to locus (for simplicity, this RNA is referred to adaptation to its natural battlefield. As a con- produce an enzyme that they called enhanced here as a guide RNA, or gRNA). The enzyme sequence, when Cas9 was brought in from the specificity Cas9 (eCas9), although the details of compares intracellular DNA to the sequence in wild and placed in human cells, it introduced their engineering and analyses differed. the gRNA, and if there is a match, Cas9 cuts the genetic changes to unintended stretches of These ‘domesticated’ Cas9 enzymes are sure invading DNA. Attackers evade detection by DNA in addition to editing the gene of inter- to be used by laboratories the world over. The changing their DNA sequence, so Cas9 evolved est7. Imagine a short-sighted witness to a crime immediate impact will be to shorten the time to cut incoming DNA even if its sequence is a attempting to identify the perpetrator in a it takes to complete a genome-editing experiless-than-perfect match to the gRNA. police line-up, relying not only on the facial ment, because the need to check for undesired Studies of this and other bacterial defence features that make the criminal unique but edits will be reduced. Cas9 has been used to mechanisms have had a major impact on also on those shared with other people, such systematically scan many human genes at a genome editing. In this process, a nuclease cuts as gender or height. This could lead to a case time for those that underlie a trait of interDNA inside the cell and, as this DNA break of mistaken identity, because a weak match to est10, and such experiments will now be more is being repaired, the desired edit (disruption, the witness’s fuzzy mental image could be rein- efficient. In agriculture — especially in species correction or insertion of a gene) takes place3–5. forced by a match on shared features. Similarly, that have extended life cycles, such as crops The first genome-editing experiments made wild-type Cas9 finds its target not only using or cattle — the use of enhanced Cas9 might use of another class of nuclease, zinc-finger the sequence-specific gRNA, but also by grasp- obviate the need to perform time-consuming nucleases (ZFNs)5, but the discovery that Cas9 ing onto the DNA backbone, which is the same crosses to obtain a pristinely edited organism. is led by a gRNA dramatically expanded the in any gene. Genome editing was first applied in the clinic FYODOR URNOV


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NEWS & VIEWS RESEARCH in 2009, when an ex vivo ZFN-based approach was used to edit certain immune cells from people with HIV11. This approach has since been used to treat more than 80 patients, and has a good safety record. Last December, the first clinical trial of in vivo gene editing, a ZFNbased approach for treating haemophilia12, passed review by the US Food and Drug Administration. ZFNs have already been engineered to a level of specificity that is comparable to that of Cas9-HF (see, and they have passed regulatory hurdles for use in clinical trials in both in vivo and ex vivo applications. The current studies inspire

confidence that the scope of clinical genome editing will continue to expand. Advances in this field offer the promise of engineering genetic cures for many diseases — a prospect that is both encouraging and within our reach. ■ Fyodor Urnov is at Sangamo BioSciences, Richmond, California 94804, USA. e-mail: [email protected] 1. 2. 3. 4.

Kleinstiver, B. P. et al. Nature 529, 490–495 (2016). Slaymaker, I. M. et al. Science 351, 84–88 (2016). Carroll, D. Annu. Rev. Biochem. 83, 409–439 (2014). Doudna, J. A. & Charpentier, E. Science 346, 1258096 (2014).


Fluorescent boost for voltage sensors The development of a voltage sensor in which a microbial rhodopsin protein is fused with a fluorescent protein enables the neuronal activity of single cells in live animals to be measured with unprecedented speed and accuracy. VIVIANA GRADINARU & N I C H O L A S C . F LY T Z A N I S


ver the past few decades, scientific discoveries have greatly deepened our understanding of biology, but our ability to understand the workings of our own minds has proved a frustrating exception. It is therefore no surprise that a major aim of neuroscience is to develop tools, in particular light-based technologies, with which to deconstruct brain function and dysfunction by controlling and recording brain activity1. Writing in Science, Gong et al.2 report one of the best tools so far for the optical monitoring of neuronal activity — a strongly fluorescent, fast-acting, voltage-sensing protein that can be used to image subcellular activity changes in model organisms. The brain uses complex electrical signalling that is orders of magnitude more efficient than the fastest computer to react to external and internal stimuli, processing these inputs and outputting behaviours. The action potentials that make up this signalling are caused by the opening and closing of protein channels in the cell membrane through which ions can flow into or out of the cell. Changes in relative ion concentrations alter the voltage across the cell membrane, causing rapid propagation of electrical currents down the length of the neuron and eventual signalling to other neurons downstream. To decode this neuronal language, we need tools that can track electrical activity from neuron to neuron across the brain. Genetically encoded calcium indicators

(GECIs)3, which fluoresce in response to the calcium-ion influx that is triggered by neuronal activity, are used as standard for tracking electrical activity in neurons in vivo. These sensor proteins can be used to monitor both population-wide and single-cell activity in chosen cell types. However, influx of

5. Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S. & Gregory, P. D. Nature Rev. Genet. 11, 636–646 (2010). 6. Bolukbasi, M. F., Gupta, A. & Wolfe, S. A. Nature Methods 13, 41–50 (2015). 7. Tsai, S. Q. et al. Nature Biotechnol. 33, 187–197 (2015). 8. Jiang, F. & Doudna, J. A. Curr. Opin. Struct. Biol. 30, 100–111 (2015). 9. Pavletich, N. P. & Pabo, C. O. Science 252, 809–817 (1991). 10. Shalem, O., Sanjana, N. E. & Zhang, F. Nature Rev. Genet. 16, 299–311 (2015). 11. Tebas, P. et al. N. Engl. J. Med. 370, 901–910 (2014). 12. Sharma, R. et al. Blood 126, 1777–1784 (2015). The author declares competing financial interests: see for details.

calcium ions is an indirect and slow measure of action potentials and cannot capture all smaller, subthreshold events — changes in membrane voltage that are not large enough to trigger an action potential, but that can nonetheless affect brain physiology. An alternative approach involves fluorescent proteins called genetically encoded voltage indicators (GEVIs), which directly report electrical activity. This approach has undergone major developments1,4 in the past few years, but, until now, could not detect fast neuronal activity in live animals. The ability to read and discriminate across the entire bandwidth of action-potential frequencies is key, because specific activities underlie distinct behaviours. Furthermore, some cells fire at high rates, and slow sensors such as GECIs might not discriminate such cells from their slower neighbours. Gong et al. developed a GEVI that can Ace


Green–yellow fluorescence


Green–yellow fluorescence

Blue–green fluorescence

Figure 1 | Sensor with a light touch.  Gong et al.2 have developed a genetically encoded voltage sensor that rapidly and accurately detects neuronal activity in live animals. The sensor is comprised of a microbial rhodopsin protein, Ace, fused to a fluorescent protein, mNeonGreen. mNeonGreen is excited by blue– green light and emits green–yellow fluorescence. Ace spans the cell membrane and absorbs green–yellow light in a voltage-dependent manner, with more light absorbed during action potentials, when the voltage increases across the membrane. Thus, the overall level of green–yellow light emitted by the fusion protein provides a readout for electrical activity, with higher light emission indicating lower membrane voltage or inactivity. (Adapted from the Gradinaru Group, California Institute of Technology.) 2 8 JA N UA RY 2 0 1 6 | VO L 5 2 9 | N AT U R E | 4 6 9

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Genome editing: The domestication of Cas9.

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