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Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacterial and Mammalian Cells via Dynamic RNA Nanotechnology

A guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. We demonstrate molecular mechanisms for both constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON → OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF → ON logic). For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to design an orthogonal library of three cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe a median conditional response of ≈4-fold for an ON → OFF “terminator switch” mechanism, ≈15-fold for an ON → OFF “splinted switch” mechanism, and ≈3-fold for an OFF → ON “toehold switch” mechanism; the median crosstalk within each cgRNA/trigger library is <2%, ≈2%, and ≈20% for the three mechanisms. To test the portability of cgRNA mechanisms prototyped in bacteria to mammalian cells, as well as to test generalizability to different effector functions, we implemented the terminator switch in HEK 293T cells expressing inducing dCas9 as the protein effector, observing a median ON → OFF conditional response of ≈4-fold with median crosstalk of ≈30% for three orthogonal cgRNA/trigger pairs. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology

Phase Distribution Manipulation through Molecular Interaction Enables Efficient Quasi-2D Perovskite Light-Emitting Diodes

Quasi-two-dimensional (quasi-2D) metal halide perovskites exhibit excellent electroluminescence (EL) performance promising for display and lighting applications, which benefits from the quantum and/or dielectric confinement effects as well as the unique energy-funneling process. However, the inefficient energy transfer caused by the intricate phase distribution with different thick nanoplatelets (n-values), as well as the existence of numerous defects, could deteriorate the EL performance of the resulting perovskite light-emitting diodes (PeLEDs). Here, a multifunctional small molecule, bistrifluoromethanesulfonimide lithium (LiTFSI), was introduced to address the above issues. The strongly electronegative fluorine atoms in LiTFSI form hydrogen bonding interactions with the ammonium heads of organic spacers, which suppress the formation of perovskite ultrathin nanoplatelet phases with small n-values, thus smoothing the energy transfer process. Meanwhile, the Lewis base sulfur oxide groups are capable of effectively passivating the uncoordinated lead ion defects and then reducing nonradiative recombination loss. Eventually, a green emission quasi-2D PeLED with an external quantum efficiency of 21.0% was achieved. This work provides a facile method to boost the performance of quasi-2D PeLEDs

Revealing a Zinc Oxide/Perovskite Luminescence Quenching Mechanism Targeting Low-Roll-off Light-Emitting Diodes

Balanced charge injection is key to achieving perovskite light-emitting diodes (PeLEDs) with a low efficiency roll-off at a high brightness. The use of zinc oxide (ZnO) with a high electron mobility as the charge transport layers is desirable; however, photoluminescence (PL) quenching of a perovskite on ZnO always occurs. Here, a quasi-two-dimensional perovskite on ZnO is explored to uncover the PL quenching mechanism, mainly ascribed to the deprotonation of ammonium cations on the ZnO film in association with the decomposition of low-dimensional perovskite phases. Surprisingly, crystal plane-dependent PL quenching results indicate that the deprotonation rate strongly correlates with the crystal orientation of the ZnO surface. We developed a strategy for suppressing perovskite PL quenching by incorporating an atomic layer deposited Al2O3 onto the ZnO film. Consequently, an efficient inverted PeLED was achieved with a maximum external quantum efficiency of 17.7% and a less discernible efficiency roll-off at a high current density

Revealing a Zinc Oxide/Perovskite Luminescence Quenching Mechanism Targeting Low-Roll-off Light-Emitting Diodes

Balanced charge injection is key to achieving perovskite light-emitting diodes (PeLEDs) with a low efficiency roll-off at a high brightness. The use of zinc oxide (ZnO) with a high electron mobility as the charge transport layers is desirable; however, photoluminescence (PL) quenching of a perovskite on ZnO always occurs. Here, a quasi-two-dimensional perovskite on ZnO is explored to uncover the PL quenching mechanism, mainly ascribed to the deprotonation of ammonium cations on the ZnO film in association with the decomposition of low-dimensional perovskite phases. Surprisingly, crystal plane-dependent PL quenching results indicate that the deprotonation rate strongly correlates with the crystal orientation of the ZnO surface. We developed a strategy for suppressing perovskite PL quenching by incorporating an atomic layer deposited Al2O3 onto the ZnO film. Consequently, an efficient inverted PeLED was achieved with a maximum external quantum efficiency of 17.7% and a less discernible efficiency roll-off at a high current density

Mask-Free Patterned Perovskite Microcavity Arrays via Inkjet Printing Targeting Laser Emission

Perovskite materials are promising candidates for the implementation of electrically pumped lasers considering the enhanced performance of perovskite-based light-emitting diodes. Nonetheless, current methods of fabricating perovskite optical microcavities require complex patterning technologies to build suitable resonant cavities for perovskite laser emission, burdening the device structure design. To address this issue, we applied inkjet printing, a maskless patterning technique, to directly create spontaneous formations of polycrystalline perovskite microcavity arrays to explore their laser-emitting action. The substrate surface tension was tuned to modulate the perovskite crystallization process in combination with optimization of printing ink recipes. As a result, polycrystalline perovskite microcavity arrays were achieved, contributing to the laser emission at 528 nm with a lasing threshold of 1.37 mJ/cm2, while simultaneously achieving high-definition patterning of flexible display. These results clearly illustrate the efficiency of inkjet printing technology in the preparation of polycrystalline perovskite optical microcavities and promote the development of flexible laser arrayed displays, providing a facile process toward the realization of perovskite-cavity laser devices

Mask-Free Patterned Perovskite Microcavity Arrays via Inkjet Printing Targeting Laser Emission

Perovskite materials are promising candidates for the implementation of electrically pumped lasers considering the enhanced performance of perovskite-based light-emitting diodes. Nonetheless, current methods of fabricating perovskite optical microcavities require complex patterning technologies to build suitable resonant cavities for perovskite laser emission, burdening the device structure design. To address this issue, we applied inkjet printing, a maskless patterning technique, to directly create spontaneous formations of polycrystalline perovskite microcavity arrays to explore their laser-emitting action. The substrate surface tension was tuned to modulate the perovskite crystallization process in combination with optimization of printing ink recipes. As a result, polycrystalline perovskite microcavity arrays were achieved, contributing to the laser emission at 528 nm with a lasing threshold of 1.37 mJ/cm2, while simultaneously achieving high-definition patterning of flexible display. These results clearly illustrate the efficiency of inkjet printing technology in the preparation of polycrystalline perovskite optical microcavities and promote the development of flexible laser arrayed displays, providing a facile process toward the realization of perovskite-cavity laser devices