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Recently developed DNA base editing methods enable the direct generation of desired point mutations in genomic DNA without generating any double-strand breaks.sup.1-3, but the issue of off-target edits has limited the application of these methods. Although several previous studies have evaluated off-target mutations in genomic DNA.sup.4-8, it is now clear that the deaminases that are integral to commonly used DNA base editors often bind to RNA.sup.9-13. For example, the cytosine deaminase APOBEC1--which is used in cytosine base editors (CBEs)--targets both DNA and RNA.sup.12, and the adenine deaminase TadA--which is used in adenine base editors (ABEs)--induces site-specific inosine formation on RNA.sup.9,11. However, any potential RNA mutations caused by DNA base editors have not been evaluated. Adeno-associated viruses are the most common delivery system for gene therapies that involve DNA editing; these viruses can sustain long-term gene expression in vivo, so the extent of potential RNA mutations induced by DNA base editors is of great concern.sup.14-16. Here we quantitatively evaluated RNA single nucleotide variations (SNVs) that were induced by CBEs or ABEs. Both the cytosine base editor BE3 and the adenine base editor ABE7.10 generated tens of thousands of off-target RNA SNVs. Subsequently, by engineering deaminases, we found that three CBE variants and one ABE variant showed a reduction in off-target RNA SNVs to the baseline while maintaining efficient DNA on-target activity. This study reveals a previously overlooked aspect of off-target effects in DNA editing and also demonstrates that such effects can be eliminated by engineering deaminases. Cytosine and adenine base editors have undesired off-target effects on RNA, but this activity can be reduced in deaminase-engineered variants while preserving on-target DNA editing., Author(s): Changyang Zhou [sup.1] [sup.2] , Yidi Sun [sup.2] [sup.3] [sup.4] , Rui Yan [sup.5] , Yajing Liu [sup.2] [sup.6] , Erwei Zuo [sup.1] [sup.7] , Chan Gu [sup.5] , [...]

We here report a genome-editing strategy to correct spinal muscular atrophy (SMA). Rather than directly targeting the pathogenic exonic mutations, our strategy employed Cas9 and guide-sgRNA for the targeted disruption of intronic splicing-regulatory elements. We disrupted intronic splicing silencers (ISSs, including ISS-N1 and ISS + 100) of survival motor neuron (SMN) 2, a key modifier gene of SMA, to enhance exon 7 inclusion and full-length SMN expression in SMA iPSCs. Survival of splicing-corrected iPSC-derived motor neurons was rescued with SMN restoration. Furthermore, co-injection of Cas9 mRNA from Streptococcus pyogenes (SpCas9) or Cas9 from Staphylococcus aureus (SaCas9) alongside their corresponding sgRNAs targeting ISS-N1 into zygotes rescued 56% and 100% of severe SMA transgenic mice (Smn −/−, SMN2 tg/−). The median survival of the resulting mice was extended to >400 days. Collectively, our study provides proof-of-principle for a new strategy to therapeutically intervene in SMA and other RNA-splicing-related diseases. [ABSTRACT FROM AUTHOR]

Low power limits the application of microbial fuel cells (MFCs). Our research mainly focuses on the modification of the electrode and looking for new anode material for high-power marine benthic microbial fuel cells(BMFCs). A MnO2/PPy composite-modified anode was fabricated by in situ chemical polymerization. Surface topography and properties were characterized by scanning electron microscopy and infrared spectroscopy, respectively, indicating that the MnO2/PPy composite is of a 'mosaic-like' microstructure. The electrochemical performance and wettability of different kinds of anode were investigated respectively. Cyclic voltammetry and linear sweep voltammetry tests show that MnO2/PPy composite-modified electrode has a typical capacitance feature; its capacitance is 3.1 times higher than that of unmodified electrode. Contact angle of the composite-modified anode reduces to 46 ± 0.5°, and its kinetic activity increased for more than 1.1 times. The maximum output power density of MnO2/PPy composite-modified cell reached 562.7 ± 10 mW m−2, which is 2.1-fold of the unmodified one. Finally, the composite-modified anode provides an alternative potential choice for high-performance cell, and the possible influence mechanism of composite materials on the BMFCs was also analyzed. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]