The combination of two techniques — optogenetics and genome editing using engineered nucleases — now provides a general means for the light-controlled regulation of any gene of interest. See Letter p.472
In this issue, Konermann et al.1 combine two sparkling biological technologies developed over the past decade. The first is optogenetics2, the process by which light-responsive proteins are engineered into target cells and used to regulate their activity. The second is the use of sequence-targeted DNA-cleaving enzymes to specifically alter the genome. By uniting these techniques, the authors present a versatile method for targeted control of gene transcription and genomic modifications.
Optogenetics can be used in cells and in living organisms, and allows cellular regulation using light of different colours, intensities and duration in a graded, non-invasive, reversible and spatiotemporally precise fashion. Most optogenetic applications so far have relied on the use of light-sensitive ion channels and ion pumps to modulate the voltage dynamically across biological membranes, in particular to elicit action potentials in neurons.
Nature offers a plethora of other processes that are regulated by light, such as those controlled by photoreceptors in plants and microorganisms. With only a few exceptions, these processes are intricately tied to their organism of origin, and their deployment in others is challenging. However, their existence suggests that optogenetics could be extended to regulating enzyme activity or could be used to induce more persistent effects by targeting DNA. Indeed, natural photoreceptors have provided design blueprints for the engineering of several biological systems with customized light responses3.
A particularly versatile strategy uses photoreceptors that associate with other proteins in a light-regulated process. In terms of performance, robustness, response time and ease of use, the blue-light-responsive protein cryptochrome 2 and its light-induced interaction with its partner protein CIB1 (ref. 4) currently have the edge over alternative photodimerizers such as the red-light-responsive phytochrome–PIF pair5. Several laboratories have successfully modulated gene transcription using photodimerizing proteins4,5,6,7,8. Initially, these optogenetic systems were directed to specific DNA sites by coupling to the DNA-binding part of the transcriptional-activator protein Gal4 (ref. 5). Light exposure recruited an interacting protein that was coupled to the activation domain of Gal4, thus initiating transcription. Although these systems are powerful7,8, they are inherently limited because they use DNA-binding domains with fixed target-sequence specificity, and because target genes have to be introduced into the host genome as exogenous DNA templates.
In parallel with the introduction of optogenetics, DNA-engineering strategies have been developed that can target unique sites among the billions of nucleotides in a genome. Early versions of such approaches9 were based on zinc-finger and transcription-activator-like effector (TALE) proteins, which contain repetitive amino-acid sequences that recognize single DNA nucleotides or nucleotide triplets, and introduce double-stranded DNA breaks on binding to these sequences. The DNA-repair process that is activated in response to this damage can be used to introduce novel genetic elements at the site. However, adjusting the sequence specificity of zinc-finger and TALE proteins entails the laborious production of customized proteins.
A more recently developed approach, called the CRISPR–Cas system10,11, overcomes this limitation. In this system, an endonuclease enzyme that induces a double-strand break is used with a sequence-specific guide RNA molecule — simply replacing the guide RNA is sufficient for sequence adaptation. The CRISPR–Cas technology stands to make engineering of zinc-finger and TALE proteins obsolete and to render genome engineering fast, efficient and inexpensive.
Capitalizing on their expertise in both optogenetics and genome engineering, Konermann et al. have overcome the sequence-restriction problem of earlier light-activated transcription-modulation approaches in their light-inducible transcriptional effector (LITE) system (Fig. 1a). The system uses a TALE protein coupled to cryptochrome 2, and CIB1 coupled to the transcriptional-activator protein VP64. This combination results in the cellular transcriptional machinery being recruited to the genomic site defined by the TALE protein when blue light is absorbed. The authors showed in vitro that, following light exposure, site-specific gene expression was enhanced by 10–20 times compared with darkness, and they convincingly validated the technology in mouse neurons and in the brains of conscious mice by monitoring light-mediated transcription of the genes Grm2 and Neurog2.
The LITE approach has several favourable characteristics. First, the light-responsive molecules of cryptochrome 2 are the chromophores flavin-adenine dinucleotide and methyltetrahydrofolate, which are universally abundant. Second, induction of transcription occurs within minutes of light exposure. Third, the response can be graded with light dose and is fully reversible after light retraction. Finally, because light can be applied non-invasively, its use is not restricted to cultured cells but extends to freely moving animals, as established for conventional optogenetic tools2.
Great power lies in the modularity and resultant versatility of this technique (Fig. 1b). By replacing constituent modules of LITE, the system can be tuned to be sensitive to light of different colours or to have different effector outputs. The authors impressively demonstrated this second possibility by interfacing LITE with various molecules that modify histones — the proteins around which DNA is wrapped. They show that their system can be used to site-specifically enhance histone methylation and acetylation — two epigenetic modifications that regulate the rate of gene transcription. The LITE approach thus enriches the optogenetic arsenal with novel applications.
Similarly, the TALE module of LITE can be exchanged for other DNA-binding modules, including ones based on the CRISPR–Cas system, as Konermann et al. demonstrate. Because the CRISPR–Cas system can be rapidly directed to different DNA sites, this will allow faster fine-tuning of the efficacy of any LITE experiments. Thus, the combination of CRISPR–Cas and LITE may truly usher in a new era of systems biology, in which gene expression and epigenetic modifications can be manipulated at the genome level with supreme sequence specificity, exquisite temporal resolution and full reversibility.
As with any new technology, there is room for improvement. In particular, it would be desirable to increase the degree of transcriptional activation by LITE. There is also the question of where the LITE system should be positioned in the genome to achieve maximum effect, but this can be easily addressed with the rapid manipulation offered by the CRISPR–Cas system. Even in its present implementation, LITE represents a powerful approach to light-controlled genome programming. Given its versatility, ease of use, performance and potential for automation, we expect this technology to be widely and rapidly taken up across many biological disciplines.