Efficient ssODN-Mediated Targeting by Avoiding Cellular Inhibitory RNAs through Precomplexed CRISPR-Cas9/sgRNA Ribonucleoprotein

CRISPR-Cas9がヒト細胞内のRNAで阻害されてしまう現象を発見し、iPS細胞での効率的な相同組み換えゲノム編集技術を実現. 京都大学プレスリリース. 2021-03-12.A simple step to enhance CRISPR-Cas9 genome editing. 京都大学プレスリリース. 2021-03-12.Combined with CRISPR-Cas9 technology and single-stranded oligodeoxynucleotides (ssODNs), specific single-nucleotide alterations can be introduced into a targeted genomic locus in induced pluripotent stem cells (iPSCs); however, ssODN knockin frequency is low compared with deletion induction. Although several Cas9 transduction methods have been reported, the biochemical behavior of CRISPR-Cas9 nuclease in mammalian cells is yet to be explored. Here, we investigated intrinsic cellular factors that affect Cas9 cleavage activity in vitro. We found that intracellular RNA, but not DNA or protein fractions, inhibits Cas9 from binding to single guide RNA (sgRNA) and reduces the enzymatic activity. To prevent this, precomplexing Cas9 and sgRNA before delivery into cells can lead to higher genome editing activity compared with Cas9 overexpression approaches. By optimizing electroporation parameters of precomplexed ribonucleoprotein and ssODN, we achieved efficiencies of single-nucleotide correction as high as 70% and loxP insertion up to 40%. Finally, we could replace the HLA-C1 allele with the C2 allele to generate histocompatibility leukocyte antigen custom-edited iPSCs

Effect of nanoscale curvature sign and bundle structure on supercritical H(2) and CH(4) adsorptivity of single wall carbon nanotube

The adsorptivities of supercritical CH(4) and H(2) of the external and internal tube walls of single wall carbon nanotube (SWCNT) were determined. The internal tube wall of the negative curvature showed the higher adsorptivities for supercritical CH(4) and H(2) than the external tube wall of the positive curvature due to their interaction potential difference. Fine SWCNT bundles were prepared by the capillary force-aided drying treatment using toluene or methanol in order to produce the interstitial pore spaces having the strongest interaction potential for CH(4) or H(2); the bundled SWCNT showed the highest adsorptivity for supercritical CH(4) and H(2). It was clearly shown that these nanostructures of SWCNTs are crucial for supercritical gas adsorptivity.ArticleADSORPTION-JOURNAL OF THE INTERNATIONAL ADSORPTION SOCIETY. 17(3):643-651 (2011)journal articl

Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping

Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond

Effect of nanoscale curvature sign and bundle structure on supercritical H2 and CH4 adsorptivity of single wall carbon nanotube

The adsorptivities of supercritical CH(4) and H(2) of the external and internal tube walls of single wall carbon nanotube (SWCNT) were determined. The internal tube wall of the negative curvature showed the higher adsorptivities for supercritical CH(4) and H(2) than the external tube wall of the positive curvature due to their interaction potential difference. Fine SWCNT bundles were prepared by the capillary force-aided drying treatment using toluene or methanol in order to produce the interstitial pore spaces having the strongest interaction potential for CH(4) or H(2); the bundled SWCNT showed the highest adsorptivity for supercritical CH(4) and H(2). It was clearly shown that these nanostructures of SWCNTs are crucial for supercritical gas adsorptivity.ArticleADSORPTION-JOURNAL OF THE INTERNATIONAL ADSORPTION SOCIETY. 17(3):643-651 (2011)journal articl

A Cryogenically Flexible Covalent Organic Framework for Efficient Hydrogen Isotope Separation by Quantum Sieving

The physical upper limit of hydrogen uptake for powder and compressed pellet MIL-101 has been experimentally investigated. Maximum uptake in pellets at 20 K achieves 9.6 wt% and 42 g L???1. Moreover, cryo-adsorption of hydrogen on pellets compared to liquid H2 possesses a larger temperature window for operation without boil-off loss, which will be beneficial for industrial applications

Highly Effective Hydrogen Isotope Separation in Nanoporous Metal-Organic Frameworks with Open Metal Sites: Direct Measurement and Theoretical Analysis

Separating gaseous mixtures that consist of very similar size is one of the critical issues in modern separation technology. Especially, the separation of the isotopes hydrogen and deuterium requires special efforts, even though these isotopes show a very large mass ratio. Conventionally, H/D separation can be realized through cryogenic distillation of the molecular species or the Girdler-sulfide process, which are among the most energy-intensive separation techniques in the chemical industry. However, costs can be significantly reduced by using highly mass-selective nanoporous sorbents. Here, we describe a hydrogen isotope separation strategy exploiting the strongly attractive open metal sites present in nanoporous metal organic frameworks of the CPO-27 family (also referred to as MOF-74). A theoretical analysis predicts an outstanding hydrogen isotopologue separation at open metal sites due to isotopal effects, which has been directly observed through cryogenic thermal desorption spectroscopy. For H-2/D-2 separation of an equimolar mixture at 60 K, the selectivity of 12 is the highest value ever measured, and this methodology shows extremely high separation efficiencies even above 77 K. Our theoretical results imply also a high selectivity for HD/H-2 separation at similar temperatures, and together with catalytically active sites, we propose a mechanism to produce D-2 from HD/H-2 mixtures with natural or enriched deuterium content