Reevaluation by the CRISPR/Cas9 knockout approach revealed that multiple pluripotency-associated lncRNAs are dispensable for pluripotency maintenance while Snora73a/b is essential for pluripotency exit

Many long noncoding RNAs(lncRNAs)have been identified through siRNA-based screening as essential regulators of embryonic stem cell(ESC)pluripotency.However,the biological and molecular functions of most lncRNAs remain unclear.Here,we employed CRISPR/Cas9-mediated knockout technology to explore the functions of 8 lncRNAs previously reported to promote pluripotency in mouse ESCs.Unexpectedly,all of these lncRNAs were dispensable for pluripotency maintenance and proliferation in mouse ESCs when disrupted individually or in combination.Single-cell transcriptomic analysis also showed that the knockout of these lncRNAs has a minimal impact on pluripotency gene expression and cell identity.We further showed that several small hairpin RNAs(shRNAs)previously used to knock down lncRNAs caused the downregulation of pluripotency genes in the corresponding lncRNA-knockout ESCs,indicating that off-target effects likely responsible for the pluripotency defects caused by these shRNAs.Interestingly,linc1343-knockout and linc1343-knockdown ESCs failed to form cystic structures and exhibited high expression of pluripotency genes during embryoid body(EB)differentiation.By reintroducing RNA products generated from the linc1343 locus,we found that two snoRNAs,Snora73a and Snora73b,but not lncRNAs,could rescue pluripotency silencing defects during EB differentiation of linc1343 knockout ESCs.Our results suggest that the 8 previously annotated pluripotency-regulating lncRNAs have no overt functions in conventional ESC culture;however,we identified snoRNA products derived from an annotated lncRNA locus as essential regulators for silencing pluripotency genes.

Efficient hepatic delivery and protein expression enabled by optimized mRNA and ionizable lipid nanoparticle

mRNA is a novel class of therapeutic modality that holds great promise in vaccination,protein replacement therapy,cancer immunotherapy,immune cell engineering etc.However,optimization of mRNA molecules and efficient in vivo delivery are quite important but challenging for its broad application.Here we present an ionizable lipid nanoparticle(iLNP)based on iBL0713 lipid for in vitro and in vivo expression of desired proteins using codon-optimized mRNAs.mRNAs encoding luciferase or erythropoietin(EPO)were prepared by in vitro transcription and formulated with proposed iLNP,to form iLP171/mRNA formulations.It was revealed that both luciferase and EPO proteins were successfully expressed by human hepatocellular carcinoma cells and hepatocytes.The maximum amount of protein expression was found at 6 h post-administration.The expression efficiency of EPO with codon-optimized mRNA was significantly higher than that of unoptimized mRNA.Moreover,no toxicity or immunogenicity was observed for these mRNA formulations.Therefore,our study provides a useful and promising platform for mRNA therapeutic development.

KLF17 promotes human naive pluripotency through repressing MAPK3 and ZIC2

The pluripotent state of embryonic stem cells(ESCs)is regulated by a sophisticated network of transcription factors.High expression of KLF17 has recently been identified as a hallmark of naive state of human ESCs(h ESCs).However,the functional role of KLF17 in naive state is not clear.Here,by employing various gain and loss-of-function approaches,we demonstrate that KLF17 is essential for the maintenance of naive state and promotes the primed to naive state transition in h ESCs.Mechanistically,we identify MAPK3 and ZIC2 as two direct targets repressed by KLF17.Overexpression of MAPK3 or ZIC2 partially blocks KLF17 from promoting the naive pluripotency.Furthermore,we find that human and mouse homologs of KLF17 retain conserved functions in promoting naive pluripotency of both species.Finally,we show that Klf17 may be essential for early embryo development in mouse.These findings demonstrate the important and conserved function of KLF17 in promoting naive pluripotency and reveal two essential transcriptional targets of KLF17 that underlie its function.

AQR is a novel type 2 diabetes-associated gene that regulates signaling pathways critical for glucose metabolism

Type 2 diabetes mellitus（T2 DM） is a common metabolic disease influenced by both genetic and environmental factors. In this study, we performed an in-house genotyping and meta-analysis study using three independent GWAS datasets of T2 DM and found that rs3743121, located 1 kb downstream of AQR,was a novel susceptibility SNP associated with T2 DM. The risk allele C of rs3743121 was correlated with the increased expression of AQR in white blood cells, similar to that observed in T2 DM models. The knockdown of AQR in HepG2 facilitated the glucose uptake, decreased the expression level of PCK2,increased the phosphorylation of GSK-3β, and restored the insulin sensitivity. Furthermore, the suppression of AQR inhibited the mTOR pathway and the protein ubiquitination process. Our study suggests that AQR is a novel type 2 diabetes-associated gene that regulates signaling pathways critical for glucose metabolism.