Curated Optogenetic Publication Database

Abstract: Organelles vitally achieve multifaceted functions to maintain cellular homeostasis. Genetic and pharmacological approaches to manipulate individual organelles are powerful in probing their physiological roles. However, many of them are either slow in action, limited to certain organelles, or rely on toxic agents. Here, we design a generalizable molecular tool utilizing phospholipase A/acyltransferases (PLAATs) for rapid defunctionalization of organelles via remodeling of the membrane phospholipids. In particular, we identify catalytically active PLAAT truncates with minimal unfavorable characteristics. Chemically-induced translocation of the optimized PLAAT to the mitochondria surface results in their rapid deformation in a phospholipase activity dependent manner, followed by loss of luminal proteins as well as dissipated membrane potential, thus invalidating the functionality. To demonstrate wide applicability, we then adapt the molecular tool in peroxisomes, and observe leakage of matrix-resident functional proteins. The technique is compatible with optogenetic control, viral delivery and operation in primary neuronal cultures. Due to such versatility, the PLAAT strategy should prove useful in studying organelle biology of diverse contexts.

Abstract: Organelles vitally achieve multifaceted functions to maintain cellular homeostasis. Genetic and pharmacological approaches to manipulate individual organelles are powerful in probing their physiological roles. However, many of them are either slow in action, limited to certain organelles, or rely on toxic agents. Here, we designed a generalizable molecular tool utilizing phospholipase A/acyltransferases (PLAATs) for rapid induction of organelle defunctionalization via remodeling of the membrane phospholipid composition. In particular, we identified a minimal, fully catalytic PLAAT with no unfavorable side effects. Chemically-induced translocation of the engineered PLAAT to the mitochondria surface resulted in their rapid deformation in a phospholipase activity dependent manner, followed by loss of luminal proteins as well as dissipated membrane potential, thus invalidating the functionality. To demonstrate wide applicability, we then adapted the molecular tool in peroxisomes, and observed leakage of matrix-resident functional proteins. The technique was compatible with optogenetic control, viral delivery and operation in primary neuronal cultures. Due to such versatility, the PLAAT strategy should present a novel utility in organelle biology of diverse contexts.

Abstract: The CRISPR-Cas9 system offers targeted genome manipulation with simplicity. Combining the CRISPR-Cas9 with optogenetics technology, we have engineered photoactivatable Cas9 to precisely control the genome sequence in a spatiotemporal manner. Here we provide a detailed protocol for optogenetic genome editing experiments using photoactivatable Cas9, including that for the generation of guide RNA vectors, light-mediated Cas9 activation, and quantification of genome editing efficiency in mammalian cells.

Abstract: The CRISPR-Cpf1 endonuclease has recently been demonstrated as a powerful tool to manipulate targeted gene sequences. Here, we performed an extensive screening of split Cpf1 fragments and identified a pair that, combined with inducible dimerization domains, enables chemical- and light-inducible genome editing in human cells. We also identified another split Cpf1 pair that is spontaneously activated. The newly generated amino and carboxyl termini of the spontaneously activated split Cpf1 can be repurposed as de novo fusion sites of artificial effector domains. Based on this finding, we generated an improved split dCpf1 activator, which has the potential to activate endogenous genes more efficiently than a previously established dCas9 activator. Finally, we showed that the split dCpf1 activator can efficiently activate target genes in mice. These results demonstrate that the present split Cpf1 provides an efficient and sophisticated genome manipulation in the fields of basic research and biotechnological applications.

Abstract: The breakthrough CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) nuclease has revolutionized our ability in genome engineering. Although Cas9 is already a powerful tool for simple and efficient target endogenous gene manipulation, further engineering of Cas9 will improve the performance of Cas9, such as gene-editing efficiency and accuracy in vivo, and expand the application possibility of this Cas9 technology. The emerging inducible Cas9 methods, which can control the activity of Cas9 using an external stimulus such as chemicals and light, have the potential to provide spatiotemporal gene manipulation in user-defined cell population at a specific time and improve the accuracy of Cas9-mediated genome editing. In this review, we focus on the recent advance in inducible Cas9 technologies, especially light-inducible Cas9, and related methodologies, and also discuss future directions of this emerging tools.

Abstract: Our improved CRISPR-Cas9-based photoactivatable transcription systems, CPTS2.0 and Split-CPTS2.0, enable high blue-light-inducible activation of endogenous target genes in various human cell lines. We achieved reversible activation of target genes with CPTS2.0 and induced neuronal differentiation in induced pluripotent stem cells (iPSCs) by upregulating NEUROD1 with Split-CPTS2.0.

Abstract: We describe an engineered photoactivatable Cas9 (paCas9) that enables optogenetic control of CRISPR-Cas9 genome editing in human cells. paCas9 consists of split Cas9 fragments and photoinducible dimerization domains named Magnets. In response to blue light irradiation, paCas9 expressed in human embryonic kidney 293T cells induces targeted genome sequence modifications through both nonhomologous end joining and homology-directed repair pathways. Genome editing activity can be switched off simply by extinguishing the light. We also demonstrate activation of paCas9 in spatial patterns determined by the sites of irradiation. Optogenetic control of targeted genome editing should facilitate improved understanding of complex gene networks and could prove useful in biomedical applications.

Abstract: Targeted endogenous gene activation is necessary for understanding complex gene networks and has great potential in medical and industrial applications. The CRISPR-Cas system offers simple and powerful tools for this purpose. However, these CRISPR-Cas-based tools for activating user-defined genes are unable to offer precise temporal control of gene expression, despite the fact that many biological phenomena are regulated by highly dynamic patterns of gene expression. Here we created a light-inducible, user-defined, endogenous gene activation system based on CRISPR-Cas9. We demonstrated that this CRISPR-Cas9-based transcription system can allow rapid and reversible targeted gene activation by light. In addition, using this system, we have exemplified photoactivation of multiple user-defined endogenous genes in mammalian cells. The present CRISPR-Cas9-based transcription system offers simple and versatile approaches for precise endogenous gene activation in basic biological research and biotechnology applications.

Abstract: Vivid (VVD) is a photoreceptor derived from Neurospora Crassa that rapidly forms a homodimer in response to blue light. Although VVD has several advantages over other photoreceptors as photoinducible homodimerization system, VVD has a critical limitation in its low dimer-forming efficiency. To overcome this limitation of wild-type VVD, here we conduct site-directed saturation mutagenesis in the homodimer interface of VVD. We have found that the Ile52Cys mutation of VVD (VVD-52C) substantially improves its homodimer-forming efficiency up to 180%. We have demonstrated the utility of VVD-52C for making a light-inducible gene expression system more robust. In addition, using VVD-52C, we have developed photoactivatable caspase-9, which enables optical control of apoptosis of mammalian cells. The present genetically engineered photoinducible homodimerization system can provide a powerful tool to optically control a broad range of molecular processes in the cell.