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Very fast CRISPR on demand Numerous efforts have been made to improve the temporal resolution of CRISPR-Cas9–mediated DNA cleavage to the hour time scale. Liu et al. developed a Cas9 system that achieved genome-editing manipulation at the second time scale (see the Perspective by Medhi and Jasin). Part of the guide RNA is chemically caged, allowing the Cas9-guide RNA complex to bind at a specific genomic locus without cleavage until activation by light. This fast CRISPR system achieves genome editing at high temporal resolution, enabling the study of early molecular events of DNA repair processes. This system also has high spatial resolution at short time scales, allowing editing of one genomic allele while leaving the other unperturbed. Science , this issue p. 1265 ; see also p. 1180

Non-membrane bound, hydrogel-like entities, such as RNA granules, nucleate essential cellular functions through their unique physico-chemical properties. However, these intracellular hydrogels have not been as extensively studied as their extracellular counterparts, primarily due to technical challenges in probing these materials in situ. Here, by taking advantage of a chemically inducible dimerization paradigm, we developed iPOLYMER, a strategy for rapid induction of protein-based hydrogels inside living cells. A series of biochemical and biophysical characterizations, in conjunction with computational modeling, revealed that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that behaves as a size-dependent molecular sieve. We studied several properties of the gel and functionalized it with RNA binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. Therefore, we here demonstrate that iPOLYMER presents a unique and powerful approach to synthetically reconstitute hydrogel-like structures including RNA granules in intact cells.

Physiological Ca2+ signalling in T lymphocytes and other cells depends on the STIM-ORAI pathway of store-operated Ca2+ entry. STIM1 and STIM2 are Ca2+ sensors located in the endoplasmic reticulum (ER) membrane, with ER-luminal domains that monitor cellular Ca2+ stores and cytoplasmic domains that gate ORAI channels in the plasma membrane. The STIM ER-luminal domain dimerizes or oligomerizes upon dissociation of Ca2+, but the mechanism transmitting activation to the STIM cytoplasmic domain has not been defined. Here we demonstrate, using Tb3+–acceptor energy transfer, that dimerization of STIM1 ER-luminal domains can initiate an extensive conformational change in murine STIM1 cytoplasmic domains. The conformational change, triggered by apposition of the predicted coiled-coil 1 (CC1) regions, releases the ORAI-activating domains from their interaction with the CC1 regions and allows physical extension of the STIM1 cytoplasmic domain across the gap between ER and plasma membrane to communicate with ORAI channels.

The rapid, inducible, reversible modulation of molecular activities by dual CID systems. Rapamycin (Rapa) treatment induces relocation of FRB-POI to the GAIs-FKBP-C2(LACT)-labeled plasma membrane and activates POI-dependent signaling event. A subsequent GA3-AM treatment induces second relocation of a whole GAIs-FKBP-C2(LACT)/rapamycin/FRB-POI complex from the plasma membrane to the Tom20-GID1-labeled mitochondria and leads to termination of POI-dependent signaling.

One fascinating recent avenue of study in the field of synthetic biology is the creation of biomolecule-based computers. The main components of a computing device consist of an arithmetic logic unit, the control unit, memory, and the input and output devices. Boolean logic gates are at the core of the operational machinery of these parts, and hence to make biocomputers a reality, biomolecular logic gates become a necessity. Indeed, with the advent of more sophisticated biological tools, both nucleic acid- and protein-based logic systems have been generated. These devices function in the context of either test tubes or living cells and yield highly specific outputs given a set of inputs. In this review, we discuss various types of biomolecular logic gates that have been synthesized, with particular emphasis on recent developments that promise increased complexity of logic gate circuitry, improved computational speed, and potential clinical applications.