Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here we develop CRISPR interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, and to dissect developmental pathways and model disease.

Fluid shear stress is intimately linked with vascular oxidative stress and atherosclerosis. We posited that atherogenic oscillatory shear stress (OSS) induced mitochondrial superoxide (mtO 2 À ) production via NADPH oxidase and c-Jun NH 2 -terminal kinase ( JNK-1 and JNK-2) signaling. In bovine aortic endothelial cells, OSS (AE3 dyn=cm 2 ) induced JNK activation, which peaked at 1 h, accompanied by an increase in fluorescein isothiocyanate-conjugated JNK fluorescent and MitoSOX Red (specific for mtO 2 À production) intensities. Pretreatment with apocynin (NADPH oxidase inhibitor) or N-acetyl cysteine (antioxidant) significantly attenuated OSS-induced JNK activation. Apocynin further reduced OSS-mediated dihydroethidium and MitoSOX Red intensities specific for cytosolic O 2 À and mtO 2 À production, respectively. As a corollary, transfecting bovine aortic endothelial cells with JNK siRNA (siJNK) and pretreating with SP600125 ( JNK inhibitor) significantly attenuated OSS-mediated mtO 2 À production. Immunohistochemistry on explants of human coronary arteries further revealed prominent phosphorylated JNK staining in OSS-exposed regions. These findings indicate that OSS induces mtO 2 À production via NADPH oxidase and JNK activation relevant for vascular oxidative stress. Antioxid. Redox Signal. 15, 1379-1388.

A method is described for the isolation of respiratory mutants of Azotobacter cinelundii with incrcascd amounts of cl-type cytochrome by selecting for the inability to reduce tctrazolium red. Five stable mutants were obtained that had six-fold higher levels of cytochrome d, increased amounts of b-type and lower amounts of o-type and c-type cytochromes than the wild-type strain. Spectral alterations in cytochrome al were also observed in the mutants. N A D H and succinate oxidase activities of membrane particles were about two fold higher in the mutants compared to the wild-type strain. Ascorbate-N,N,N',N'-tetramethyl-p-phenylene diaminc oxidase activity was barely detectable in membranc particles of the mutants. These results are consistent with an increasc in the amount of the cytochrome d oxidase branch and a decrease in the amount and activity of the cytochrome 0, a1 oxidase branch in the mutants. Growth rates under oxygen-excess conditions and respiratory-linked proton translocation ratios of the mutant and wild-type strains were similar as were the photochemical spectral and kinetic properties of cytochrome d.The respiratory chain of Azotobacter vinelundii has been extensively studied [l, 21. The components of the respiratory chain include highly active flavin-dependent NADH and malate dehydrogenases, hydrogenase, ubiquinone-8, and at least seven spectroscopically detectable cytochromes, b-560. [3,9: 141. Sites of cncrgy conservation occur at the level of NADH dchydrogenase, hydrogenase, the ubiquinone region [4,9,7,16,17,33]. Recent studies diminish the possibility that the cytochrome c-al, o branch is involved in the energy conservation cfficicncy under non-oxygen-limiting growth conditions [11,12.13:15].In addition to its role in energy conservation, it has been proposed that the high activity of the respiratory chain protects the oxygen-sensitive nitrogenase complex in Azotohacter from inactivation [18 -201. Respiratory protection of nitrogenase at higher dissolved oxygen tensions is provided by an increase in whole cell respiration rate which is accompanied by increases in N A D H oxidase activity and levels of cytochrome d [18 -201. The study of respiratory mutants of A . uinelandii has proven to be useful in determining the contribution of each branch of the respiratory chain in the oxidation of physiological substrates and in the efficiency of encrgy conservation. Biochemical studies of mutants unable to reoxidize Ph(NMe,), could not demonstrate a significant role for the MATbRIALS A N D METHODS Bacterial strainsThe wild-type strain AVOP of Azobacter rinelandii was used. Strains AV90 and AVZ 07 are Ph(NMe,),-oxidasenegative mutants of AVOP [I1 -131 (Hoffman and Dcr Vartanian, unpublished results). Mt.dia and conditions of cultiuatiorzEach strain was routinely grown in 1.5 1 of modified Burk's medium [21] with 2 '4 sucrose (wiv) without an added nitrogen source. Other ctrbon sourccs were added at a final concentration of 0.2 ,d (wlv) and nitrogen sources were added to givc a final combined niiro...

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Azotobacter vinelandii strain AVOP (wild type) and an ascorbate-N,N,N',N'-tetramethylene-p-phenylenediamine oxidase-negative mutant (AV11) were each grown in O2-limited chemostat cultures. The results showed that the mutant strain grew and used O2 less efficiently than the wild-type strain. Respiration rates of membrane particles with NADH or malate as the substrate were similar for each strain. Succinate oxidase activity was about fourfold lower in membrane particles prepared from mutant than from wild-type strain. Cyanide at a concentration that completely inhibited ascorbate-TMPD oxidase activity resulted in a 50% inhibition of NADH oxidase activity in membrane particles of AVOP. These data suggest that the cytochrome o, a1, oxidase branch of the respiratory chain may be important in the physiology of A. vinelandii under O2-limiting growth conditions.