Improvement of TaC9-ABE mediated correction of human SMN2 gene.

Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Gene editing technology repairs the conversion of the 6th base T to C in exon 7 of the paralogous SMN2 gene, compensating for the SMN protein expression and promoting the survival and function of motor neurons. However, low editing efficiency and unintended off-target effects limit the application of this technology. Here, we optimized a TaC9-adenine base editor (ABE) system by combining Cas9 nickase with the transcription activator-like effector (TALE)-adenosine deaminase fusion protein to effectively and precisely edit SMN2 without detectable Cas9 dependent off-target effects in human cell lines. We also generated human SMA-induced pluripotent stem cells (SMA-iPSCs) through the mutation of the splice acceptor or deletion of the exon 7 of SMN1. TaC9-R10 induced 45% SMN2 T6 > C conversion in the SMA-iPSCs. The SMN2 T6 > C splice-corrected SMA-iPSCs were directionally differentiated into motor neurons, exhibiting SMN protein recovery and antiapoptosis ability. Therefore, the TaC9-ABE system with dual guides from the combination of Cas9 with TALE could be a potential therapeutic strategy for SMA with high efficacy and safety.

[Rapamycin mediated caspase 9 homodimerization to safeguard human pluripotent stem cell therapy].

Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 (riC9) gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.

A 3D structure C/Si/ZnCo2O4/CC anode for flexible lithium-ion batteries with high capacity and fast charging ability.

ZnCo2O4 has attracted extensive attention as a bimetallic transition metal oxide anode material for lithium-ion batteries (LIBs) with high capacity. However, there is still a long way to go to meet the increasing demand for commercial batteries due to their modest conductivity and unobtrusive cycling stability. The use of finely controlled nanostructures and combination with other anode materials are the two main ways to improve the battery performance of ZnCo2O4. Herein, ZnCo2O4 (ZCO) nanosheets were in situ grown on carbon cloth (CC) through a facile solution method. Si was coated onto the ZCO nanosheet arrays by the magnetron sputtering method (SCZO/CC) to acheive the capacity increase. A layer of C was further coated onto SZCO/CC to improve the electrical conductivity of the whole electrode and to protect the SZCO nanostructure. The obtained CSZCO/CC electrode exhibits a high reversible areal capacity of 1.16 mA h cm-2 at 5 mA cm-2 after 500 cycles. At an ultra-high current density of 10 mA cm-2, the CSZCO/CC electrode can still present a capacity of 0.38 mA h cm-2 and maintain a capacity retention of 88.4% for 2000 cycles. In situ Raman spectroscopy was used to study the relationship between the electrochemical performance and structure of the electrode materials. The carbon cloth was found to have contributed a nonnegligible part of the capacity of the electrode.