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 go.nature.com/mkl6v1), 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).
NE URO SCIENCE
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
O
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 go.nature.com/vvxzp6 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
Membrane
Green–yellow fluorescence
mNeonGreen
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
© 2016 Macmillan Publishers Limited. All rights reserved
RESEARCH NEWS & VIEWS monitor fast neuronal activity in single cells in live flies and mice. The authors used a microbial rhodopsin protein called Ace as the basis for their voltage sensor. Microbial rhodopsins are light-sensitive ion channels, and were initially adopted in neuroscience for their ability to generate electrical currents and so to modulate neuronal activity1. More recently, these proteins have been used to monitor electrical currents because they fluoresce in a voltage-dependent manner5. They are fast and sensitive sensors, but their use in live organisms6 has been hampered by the fact that they fluoresce only weakly. The researchers bypassed this obstacle by fusing Ace with the fluorescent protein mNeonGreen. In this configuration, blue– green light excites mNeonGreen, which emits green–yellow fluorescence. A portion of this fluorescence is absorbed in a voltage-dependent manner by Ace, causing mNeonGreen-emitted fluorescence to decrease as the membrane voltage rises and neuronal activity increases, and to increase as the membrane voltage falls (Fig. 1). In vitro, the Ace–mNeon fusion protein acts six times faster and can resolve closely spaced, repeating action potentials much more accurately than similar protein fusions7. To assess the capabilities of their tool in vivo, Gong and colleagues compared it with GECIs in live mice and flies. Measurements taken using Ace–mNeon during a visual task corroborated previous measurements taken with GECIs. In mice, Ace–mNeon flawlessly reported single action potentials in neurons at the surface of the brain’s cortex region, 20 times faster than is possible using GECIs. This is an impressive achievement, because intact mammalian tissue is opaque and can be naturally fluorescent — both of which are factors that can mask the signal from fluorescent proteins. In flies, Ace–mNeon recorded more than 18,000 action potentials with perfect accuracy, and detected odour-evoked subthreshold and fast voltage changes that a GECI failed to pick up. Furthermore, the authors used the protein to track voltage propagation from one side of a cell to the other with submillisecond precision. Such precision tracking was previously unachievable in live flies. Although the sensor’s performance is impressive, major challenges remain before it can replace GECIs in vivo. First, the authors used conventional fluorescence microscopy for in vivo imaging. The effectiveness of this type of imaging for sensor detection relies on sparse expression of Ace–mNeon, limiting the number of cells that can be imaged concurrently. Second, for maximum impact, a fast sensor requires fast imaging, but imaging speed and field of view are inversely correlated in current imaging techniques, so rapid imaging limits the ability to simultaneously investigate many cells. The combination of fluorescence micro scopy and limited field of view meant that Gong et al. could study only a handful of cells
at a time. A third challenge is that, although mNeonGreen is three times more stable to light than other rhodopsin-paired fluorescent proteins, extended continuous imaging sessions still ‘bleach’ the protein, decreasing its fluorescence. This limitation could be bypassed by using multiple short exposures, or by spacing measurements widely enough for protein turnover to replace the photobleached sensors. The benefits of using GEVIs such as Ace–mNeon to image activity in live animals are undeniable. Nonetheless, better hardware is required to realize the full potential of these voltage reporters. Until that is available, calcium sensors will remain the gold standard for studying densely labelled cell populations simultaneously over extended imaging sessions, especially in deep brain areas. The development of technologies such as microendoscopy8 and fibre photometry9 has enabled calcium imaging of subcortical brain regions, and fine-tuning these techniques for use with GEVIs is an exciting possibility for the future. Overall, Gong and colleagues’ study
highlights the power of microbial rhodopsins, especially when paired with strongly fluor escent proteins, and the need for continued development of these tools hand-in-hand with microscopy techniques. ■ Viviana Gradinaru and Nicholas C. Flytzanis are in the Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA. e-mail: [email protected] 1. Emiliani, V., Cohen, A. E., Deisseroth, K. & Hausser, M. J. Neurosci. 35, 13917–13926 (2015). 2. Gong, Y. et al. Science 350, 1361–1366 (2015). 3. Chen, T.-W. et al. Nature 499, 295–300 (2013). 4. Knöpfel, T., Gallero-Salas, Y. & Song, C. Curr. Opin. Chem. Biol. 27, 75–83 (2015). 5. Kralj, J. M., Douglass, A. D., Hochbaum, D. R., Maclaurin, D. & Cohen, A. E. Nature Meth. 9, 90–95 (2012). 6. Flytzanis, N. C. et al. Nature Commun. 5, 4894 (2014). 7. Gong, Y., Wagner, M. J., Li, J. Z. & Schnitzer, M. J. Nature Commun. 5, 3674 (2014). 8. Lecoq, J. et al. Nature Neurosci. 17, 1825–1829 (2014). 9. Lerner, T. N. et al. Cell 162, 635–647 (2015).
EVO LU TI O N
A lizard that generates heat Birds and mammals generate heat to regulate body temperature, but most non-avian reptiles cannot. The discovery of endothermy during the reproductive period of a tegu lizard sheds light on the evolution of this characteristic. C O L L E E N G . FA R M E R
T
he avian and mammalian lineages diverged 320 million years ago, and since that time both lineages have converged on a radically different approach to life from that of their common ancestor. Birds and mammals are endotherms, meaning they use internal heat to regulate their body temperature; their ancestor, and many extant animals such as amphibians and non-avian reptiles, are ectotherms that rely on external heat sources (Fig. 1). Understanding the convergent evolution of endothermy in birds and mammals is a central question in evolutionary physiology, because thermal biology is linked to fundamental traits such as body size, food requirements and aspects of reproduction. Writing in Science Advances, Tattersall et al.1 report the remarkable discovery that a lizard species uses endothermy during its reproductive period. Their finding supports the idea2,3 that the ability to exert control over temperature during reproduction was the common selective agent that drove the evolution of
4 7 0 | N AT U R E | VO L 5 2 9 | 2 8 JA N UA RY 2 0 1 6
© 2016 Macmillan Publishers Limited. All rights reserved
endothermy in birds and mammals. The transition from aquatic to terrestrial habitats presented animals with new challenges to reproduction; chief among these was the fact that eggs laid on land are at risk of desiccation and are subject to greater fluctuations in temperature than eggs laid in water. One lineage of animals — the amniotes — evolved eggs containing a series of fluid-filled membranes, which reduced the risk of desiccation (Fig. 1). Many amniotes further evolved an ability to exert control over temperature during reproduction. For example, viviparity (giving birth to live young rather than laying eggs) allows females to control developmental temperature by gaining heat through basking, and has evolved independently more than 100 times in lizards and snakes4. Tattersall et al. studied black and white tegu lizards (Salvator merianae), which inhabit tropical, subtropical and temperate climates throughout the plains east of the Andes Mountains. During autumn and winter, the lizards hibernate in burrows, after which their reproductive phase begins. Males undergo a surge in
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).
NE URO SCIENCE
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
O
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 go.nature.com/vvxzp6 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
Membrane
Green–yellow fluorescence
mNeonGreen
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
© 2016 Macmillan Publishers Limited. All rights reserved
RESEARCH NEWS & VIEWS monitor fast neuronal activity in single cells in live flies and mice. The authors used a microbial rhodopsin protein called Ace as the basis for their voltage sensor. Microbial rhodopsins are light-sensitive ion channels, and were initially adopted in neuroscience for their ability to generate electrical currents and so to modulate neuronal activity1. More recently, these proteins have been used to monitor electrical currents because they fluoresce in a voltage-dependent manner5. They are fast and sensitive sensors, but their use in live organisms6 has been hampered by the fact that they fluoresce only weakly. The researchers bypassed this obstacle by fusing Ace with the fluorescent protein mNeonGreen. In this configuration, blue– green light excites mNeonGreen, which emits green–yellow fluorescence. A portion of this fluorescence is absorbed in a voltage-dependent manner by Ace, causing mNeonGreen-emitted fluorescence to decrease as the membrane voltage rises and neuronal activity increases, and to increase as the membrane voltage falls (Fig. 1). In vitro, the Ace–mNeon fusion protein acts six times faster and can resolve closely spaced, repeating action potentials much more accurately than similar protein fusions7. To assess the capabilities of their tool in vivo, Gong and colleagues compared it with GECIs in live mice and flies. Measurements taken using Ace–mNeon during a visual task corroborated previous measurements taken with GECIs. In mice, Ace–mNeon flawlessly reported single action potentials in neurons at the surface of the brain’s cortex region, 20 times faster than is possible using GECIs. This is an impressive achievement, because intact mammalian tissue is opaque and can be naturally fluorescent — both of which are factors that can mask the signal from fluorescent proteins. In flies, Ace–mNeon recorded more than 18,000 action potentials with perfect accuracy, and detected odour-evoked subthreshold and fast voltage changes that a GECI failed to pick up. Furthermore, the authors used the protein to track voltage propagation from one side of a cell to the other with submillisecond precision. Such precision tracking was previously unachievable in live flies. Although the sensor’s performance is impressive, major challenges remain before it can replace GECIs in vivo. First, the authors used conventional fluorescence microscopy for in vivo imaging. The effectiveness of this type of imaging for sensor detection relies on sparse expression of Ace–mNeon, limiting the number of cells that can be imaged concurrently. Second, for maximum impact, a fast sensor requires fast imaging, but imaging speed and field of view are inversely correlated in current imaging techniques, so rapid imaging limits the ability to simultaneously investigate many cells. The combination of fluorescence micro scopy and limited field of view meant that Gong et al. could study only a handful of cells
at a time. A third challenge is that, although mNeonGreen is three times more stable to light than other rhodopsin-paired fluorescent proteins, extended continuous imaging sessions still ‘bleach’ the protein, decreasing its fluorescence. This limitation could be bypassed by using multiple short exposures, or by spacing measurements widely enough for protein turnover to replace the photobleached sensors. The benefits of using GEVIs such as Ace–mNeon to image activity in live animals are undeniable. Nonetheless, better hardware is required to realize the full potential of these voltage reporters. Until that is available, calcium sensors will remain the gold standard for studying densely labelled cell populations simultaneously over extended imaging sessions, especially in deep brain areas. The development of technologies such as microendoscopy8 and fibre photometry9 has enabled calcium imaging of subcortical brain regions, and fine-tuning these techniques for use with GEVIs is an exciting possibility for the future. Overall, Gong and colleagues’ study
highlights the power of microbial rhodopsins, especially when paired with strongly fluor escent proteins, and the need for continued development of these tools hand-in-hand with microscopy techniques. ■ Viviana Gradinaru and Nicholas C. Flytzanis are in the Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA. e-mail: [email protected] 1. Emiliani, V., Cohen, A. E., Deisseroth, K. & Hausser, M. J. Neurosci. 35, 13917–13926 (2015). 2. Gong, Y. et al. Science 350, 1361–1366 (2015). 3. Chen, T.-W. et al. Nature 499, 295–300 (2013). 4. Knöpfel, T., Gallero-Salas, Y. & Song, C. Curr. Opin. Chem. Biol. 27, 75–83 (2015). 5. Kralj, J. M., Douglass, A. D., Hochbaum, D. R., Maclaurin, D. & Cohen, A. E. Nature Meth. 9, 90–95 (2012). 6. Flytzanis, N. C. et al. Nature Commun. 5, 4894 (2014). 7. Gong, Y., Wagner, M. J., Li, J. Z. & Schnitzer, M. J. Nature Commun. 5, 3674 (2014). 8. Lecoq, J. et al. Nature Neurosci. 17, 1825–1829 (2014). 9. Lerner, T. N. et al. Cell 162, 635–647 (2015).
EVO LU TI O N
A lizard that generates heat Birds and mammals generate heat to regulate body temperature, but most non-avian reptiles cannot. The discovery of endothermy during the reproductive period of a tegu lizard sheds light on the evolution of this characteristic. C O L L E E N G . FA R M E R
T
he avian and mammalian lineages diverged 320 million years ago, and since that time both lineages have converged on a radically different approach to life from that of their common ancestor. Birds and mammals are endotherms, meaning they use internal heat to regulate their body temperature; their ancestor, and many extant animals such as amphibians and non-avian reptiles, are ectotherms that rely on external heat sources (Fig. 1). Understanding the convergent evolution of endothermy in birds and mammals is a central question in evolutionary physiology, because thermal biology is linked to fundamental traits such as body size, food requirements and aspects of reproduction. Writing in Science Advances, Tattersall et al.1 report the remarkable discovery that a lizard species uses endothermy during its reproductive period. Their finding supports the idea2,3 that the ability to exert control over temperature during reproduction was the common selective agent that drove the evolution of
4 7 0 | N AT U R E | VO L 5 2 9 | 2 8 JA N UA RY 2 0 1 6
© 2016 Macmillan Publishers Limited. All rights reserved
endothermy in birds and mammals. The transition from aquatic to terrestrial habitats presented animals with new challenges to reproduction; chief among these was the fact that eggs laid on land are at risk of desiccation and are subject to greater fluctuations in temperature than eggs laid in water. One lineage of animals — the amniotes — evolved eggs containing a series of fluid-filled membranes, which reduced the risk of desiccation (Fig. 1). Many amniotes further evolved an ability to exert control over temperature during reproduction. For example, viviparity (giving birth to live young rather than laying eggs) allows females to control developmental temperature by gaining heat through basking, and has evolved independently more than 100 times in lizards and snakes4. Tattersall et al. studied black and white tegu lizards (Salvator merianae), which inhabit tropical, subtropical and temperate climates throughout the plains east of the Andes Mountains. During autumn and winter, the lizards hibernate in burrows, after which their reproductive phase begins. Males undergo a surge in
