Development of deaminase-free T-to-S base editor and C-to-G base editor by engineered human uracil DNA glycosylase.

DNA base editors enable direct editing of adenine (A), cytosine (C), or guanine (G), but there is no base editor for direct thymine (T) editing currently. Here we develop two deaminase-free glycosylase-based base editors for direct T editing (gTBE) and C editing (gCBE) by fusing Cas9 nickase (nCas9) with engineered human uracil DNA glycosylase (UNG) variants. By several rounds of structure-informed rational mutagenesis on UNG in cultured human cells, we obtain gTBE and gCBE with high activity of T-to-S (i.e., T-to-C or T-to-G) and C-to-G conversions, respectively. Furthermore, we conduct parallel comparison of gTBE/gCBE with those recently developed using other protein engineering strategies, and find gTBE/gCBE show the outperformance. Thus, we provide several base editors, gTBEs and gCBEs, with corresponding engineered UNG variants, broadening the targeting scope of base editors.

Programmable deaminase-free base editors for G-to-Y conversion by engineered glycosylase.

Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing is available at present. Here we developed a deaminase-free glycosylase-based guanine base editor (gGBE) with G editing ability, by fusing Cas9 nickase with engineered N-methylpurine DNA glycosylase protein (MPG). By several rounds of MPG mutagenesis via unbiased and rational screening using an intron-split EGFP reporter, we demonstrated that gGBE with engineered MPG could increase G editing efficiency by more than 1500 fold. Furthermore, this gGBE exhibited high base editing efficiency (up to 81.2%) and high G-to-T or G-to-C (i.e. G-to-Y) conversion ratio (up to 0.95) in both cultured human cells and mouse embryos. Thus, we have provided a proof-of-concept of a new base editing approach by endowing the engineered DNA glycosylase the capability to selectively excise a new type of substrate.

Programmable A-to-Y base editing by fusing an adenine base editor with an N-methylpurine DNA glycosylase.

Here we developed an adenine transversion base editor, AYBE, for A-to-C and A-to-T transversion editing in mammalian cells by fusing an adenine base editor (ABE) with hypoxanthine excision protein N-methylpurine DNA glycosylase (MPG). We also engineered AYBE variants enabling targeted editing at genomic loci with higher transversion editing activity (up to 72% for A-to-C or A-to-T editing).

High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects.

CRISPR-Cas13 systems have recently been used for targeted RNA degradation in various organisms. However, collateral degradation of bystander RNAs has limited their in vivo applications. Here, we design a dual-fluorescence reporter system for detecting collateral effects and screening Cas13 variants in mammalian cells. Among over 200 engineered variants, several Cas13 variants including Cas13d and Cas13X exhibit efficient on-target activity but markedly reduced collateral activity. Furthermore, transcriptome-wide off-targets and cell growth arrest induced by Cas13 are absent for these variants. High-fidelity Cas13 variants show similar RNA knockdown activity to wild-type Cas13 but no detectable collateral damage in transgenic mice or adeno-associated-virus-mediated somatic cell targeting. Thus, high-fidelity Cas13 variants with minimal collateral effects are now available for targeted degradation of RNAs in basic research and therapeutic applications.

Effects of Different Types of Fibers on the Physical and Mechanical Properties of MICP-Treated Calcareous Sand.

Microbial-induced calcite precipitation (MICP) has been a promising method to improve geotechnical engineering properties through the precipitation of calcium carbonate (CaCO3) on the contact and surface of soil particles in recent years. In the present experiment, water absorption and unconfined compressive strength (UCS) tests were carried out to investigate the effects of three different fiber types (glass fiber, polyester fiber, and hemp fiber) on the physical and mechanical properties of MICP-treated calcareous sand. The fibers used were at 0%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, and 0.40% relative to the weight of the sand. The results showed that the failure strain and ductility of the samples could be improved by adding fibers. Compared to biocemented sand (BS), the water absorption of these three fiber-reinforced biocemented sands were, respectively, decreased by 11.60%, 21.18%, and 7.29%. UCS was, respectively, increased by 24.20%, 60.76%, and 6.40%. Polyester fiber produced the best effect, followed by glass fiber and hemp fiber. The optimum contents of glass fiber and polyester fiber were 0.20% and 0.25%, respectively. The optimum content of hemp fiber was within the range of 0.20-0.25%. Light-emitting diode (LED) microscope and scanning electron microscope (SEM) images lead to the conclusion that only a little calcite precipitation had occurred around the hemp fiber, leading to a poor bonding effect compared to the glass and polyester fibers. It was therefore suggested that polyester fiber should be used to improve the properties of biocemented sand.

Fbxl4 Serves as a Clock Output Molecule that Regulates Sleep through Promotion of Rhythmic Degradation of the GABAA Receptor.

The timing of sleep is tightly governed by the circadian clock, which contains a negative transcriptional feedback loop and synchronizes the physiology and behavior of most animals to daily environmental oscillations. However, how the circadian clock determines the timing of sleep is largely unclear. In vertebrates and invertebrates, the status of sleep and wakefulness is modulated by the electrical activity of pacemaker neurons that are circadian regulated and suppressed by inhibitory GABAergic inputs. Here, we showed that Drosophila GABAA receptors undergo rhythmic degradation in arousal-promoting large ventral lateral neurons (lLNvs) and their expression level in lLNvs displays a daily oscillation. We also demonstrated that the E3 ligase Fbxl4 promotes GABAA receptor ubiquitination and degradation and revealed that the transcription of fbxl4 in lLNvs is CLOCK dependent. Finally, we demonstrated that Fbxl4 regulates the timing of sleep through rhythmically reducing GABA sensitivity to modulate the excitability of lLNvs. Our study uncovered a critical molecular linkage between the circadian clock and the electrical activity of pacemaker neurons and demonstrated that CLOCK-dependent Fbxl4 expression rhythmically downregulates GABAA receptor level to increase the activity of pacemaker neurons and promote wakefulness.

Neurexin regulates nighttime sleep by modulating synaptic transmission.

Neurexins are cell adhesion molecules involved in synaptic formation and synaptic transmission. Mutations in neurexin genes are linked to autism spectrum disorders (ASDs), which are frequently associated with sleep problems. However, the role of neurexin-mediated synaptic transmission in sleep regulation is unclear. Here, we show that lack of the Drosophila α-neurexin homolog significantly reduces the quantity and quality of nighttime sleep and impairs sleep homeostasis. We report that neurexin expression in Drosophila mushroom body (MB) αß neurons is essential for nighttime sleep. We demonstrate that reduced nighttime sleep in neurexin mutants is due to impaired αß neuronal output, and show that neurexin functionally couples calcium channels (Cac) to regulate synaptic transmission. Finally, we determine that αß surface (αßs) neurons release both acetylcholine and short neuropeptide F (sNPF), whereas αß core (αßc) neurons release sNPF to promote nighttime sleep. Our findings reveal that neurexin regulates nighttime sleep by mediating the synaptic transmission of αß neurons. This study elucidates the role of synaptic transmission in sleep regulation, and might offer insights into the mechanism of sleep disturbances in patients with autism disorders.

Drosophila neuroligin 4 regulates sleep through modulating GABA transmission.

Sleep is an essential and evolutionarily conserved behavior that is closely related to synaptic function. However, whether neuroligins (Nlgs), which are cell adhesion molecules involved in synapse formation and synaptic transmission, are involved in sleep is not clear. Here, we show that Drosophila Nlg4 (DNlg4) is highly expressed in large ventral lateral clock neurons (l-LNvs) and that l-LNv-derived DNlg4 is essential for sleep regulation. GABA transmission is impaired in mutant l-LNv, and sleep defects in dnlg4 mutant flies can be rescued by genetic manipulation of GABA transmission. Furthermore, dnlg4 mutant flies exhibit a severe reduction in GABAA receptor RDL clustering, and DNlg4 associates with RDLs in vivo. These results demonstrate that DNlg4 regulates sleep through modulating GABA transmission in l-LNvs, which provides the first known link between a synaptic adhesion molecule and sleep in Drosophila.

Phototransduction in Drosophila.

The Drosophila visual transduction is the fastest known G protein-coupled signaling cascade and has been served as a model for understanding the molecular mechanisms of other G protein-coupled signaling cascades. Numbers of components in visual transduction machinery have been identified. Based on the functional characterization of these genes, a model for Drosophila phototransduction has been outlined, including rhodopsin activation, phosphoinoside signaling, and the opening of TRP and TRPL channels. Recently, the characterization of mutants, showing slow termination, revealed the physiological significance and the mechanism of rapid termination of light response.