Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs

Summary: We describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function: TTN, MYL7, MYL2, TNNI1, and ACTN2. CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis. : Gunawardane and colleagues use CRISPR/Cas9 to deliver an excisable cassette to transcriptionally silent loci in hiPSCs, then accomplish excision of the cassette in a second step utilizing Cas9/CRISPR and the MMEJ and NHEJ DNA-repair pathways. Excision results in mEGFP tagging of the targeted loci. Upon differentiation, each of five tagged cell lines appropriately expresses a unique fluorescent fusion protein localized to the sarcomere in live cardiomyocytes. Keywords: CRISPR/Cas9, genome editing, cardiomyocyte differentiation, stem cells, iPSCs, MMEJ, live imaging, endogenous fluorescent tagging, mEGFP, HD

Crustal and time-varying magnetic fields at the InSight landing site on Mars

International audienceMagnetic fields provide a window into a planet’s interior structure and evolution, including its atmospheric and space environments. Satellites at Mars have measured crustal magnetic fields indicating an ancient dynamo. These crustal fields interact with the solar wind to generate transient fields and electric currents in Mars’s upper atmosphere. Surface magnetic field data play a key role in understanding these effects and the dynamo. Here we report measurements of magnetic field strength and direction at the InSight (Interior Exploration using Seismic Investigations, Geodesy and Heat Transport) landing site on Mars. We find that the field is ten times stronger than predicted by satellite-based models. We infer magnetized rocks beneath the surface, within ~150 km of the landing site, consistent with a past dynamo with Earth-like strength. Geological mapping and InSight seismic data suggest that much or all of the magnetization sources are carried in basement rocks, which are at least 3.9 billion years old and are overlain by between 200 m and ~10 km of lava flows and modified ancient terrain. Daily variations in the magnetic field indicate contributions from ionospheric currents at 120 km to 180 km altitude. Higher-frequency variations are also observed; their origin is unknown, but they probably propagate from even higher altitudes to the surface. We propose that the time-varying fields can be used to investigate the electrical conductivity structure of the martian interio