CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes.

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

Human neurons lacking amyloid precursor protein exhibit cholesterol-associated developmental and presynaptic deficits.

Amyloid precursor protein (APP) produces aggregable ß-amyloid peptides and its mutations are associated with familial Alzheimer's disease (AD), which makes it one of the most studied proteins. However, APP's role in the human brain remains unclear despite years of investigation. One problem is that most studies on APP have been carried out in cell lines or model organisms, which are physiologically different from human neurons in the brain. Recently, human-induced neurons (hiNs) derived from induced pluripotent stem cells (iPSCs) provide a practical platform for studying the human brain in vitro. Here, we generated APP-null iPSCs using CRISPR/Cas9 genome editing technology and differentiate them into matured human neurons with functional synapses using a two-step procedure. During hiN differentiation and maturation, APP-null cells exhibited less neurite growth and reduced synaptogenesis in serum-free but not serum-containing media. We have found that cholesterol (Chol) remedies those developmental defects in APP-null cells, consistent with Chol's role in neurodevelopment and synaptogenesis. The phenotypic rescue was also achieved by coculturing those cells with wild-type mouse astrocytes, suggesting that APP's developmental role is likely astrocytic. Next, we examined matured hiNs using patch-clamp recording and detected reduced synaptic transmission in APP-null cells. This change was largely due to decreased synaptic vesicle (SV) release and retrieval, which was confirmed by live-cell imaging using two SV-specific fluorescent reporters. Adding Chol shortly before stimulation mitigated the SV deficits in APP-null iNs, indicating that APP facilitates presynaptic membrane Chol turnover during the SV exo-/endocytosis cycle. Taken together, our study in hiNs supports the notion that APP contributes to neurodevelopment, synaptogenesis, and neurotransmission via maintaining brain Chol homeostasis. Given the vital role of Chol in the central nervous system, the functional connection between APP and Chol bears important implications in the pathogenesis of AD.

The assembly of mammalian SWI/SNF chromatin remodeling complexes is regulated by lysine-methylation dependent proteolysis.

The assembly of mammalian SWI/SNF chromatin remodeling complexes is developmentally programed, and loss/mutations of SWI/SNF subunits alter the levels of other components through proteolysis, causing cancers. Here, we show that mouse Lsd1/Kdm1a deletion causes dramatic dissolution of SWI/SNF complexes and that LSD1 demethylates the methylated lysine residues in SMARCC1 and SMARCC2 to preserve the structural integrity of SWI/SNF complexes. The methylated SMARCC1/SMARCC2 are targeted for proteolysis by L3MBTL3 and the CRL4DCAF5 ubiquitin ligase complex. We identify SMARCC1 as the critical target of LSD1 and L3MBTL3 to maintain the pluripotency and self-renewal of embryonic stem cells. L3MBTL3 also regulates SMARCC1/SMARCC2 proteolysis induced by the loss of SWI/SNF subunits. Consistently, mouse L3mbtl3 deletion causes striking accumulation of SWI/SNF components, associated with embryonic lethality. Our studies reveal that the assembly/disassembly of SWI/SNF complexes is dynamically controlled by a lysine-methylation dependent proteolytic mechanism to maintain the integrity of the SWI/SNF complexes.

The water relations and xylem attributes of albino redwood shoots (Sequioa sempervirens (D. Don.) Endl.).

Plants that lack chlorophyll are rare and typically restricted to holoparasites that obtain their carbon, water and mineral resources from a host plant. Although not parasites in the traditional sense, albino foliage, such as the sprouts that sometimes develop from redwood tree trunks, are comparable in function. They occur sporadically, and can reach the size of shrubs and in rare cases, trees. Albino redwoods are interesting because in addition to their reduced carbon resources, the absence of chloroplasts may impede proper stomatal function, and both aspects may have upstream consequences on water transport and xylem quality. We examined the water relations, water transport and xylem anatomical attributes of albino redwoods and show that similar to achlorophyllous and parasitic plants, albino redwoods have notably higher stomatal conductance than green sprouts. Given that stem xylem tracheid size as well as water transport efficiency are nearly equivalent in both albino and green individuals, we attribute the increased leaf water loss in albino sprouts to lower leaf to xylem area ratios, which favour improved hydration relative to green sprouts. The stems of albino redwoods were more vulnerable to drought-induced embolism than green stems, and this was consistent with the albino's weaker tracheids, as characterized by wall thickness to lumen diameter measures. Our results are both complementary and consistent with previous research on achlorophyllous plants, and suggest that the loss of stomatal control and photosynthetic capacity results in substantial vascular and anatomical adjustments.