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1. Towards a plant model for enigmatic U‐to‐C RNA editing: the organelle genomes, transcriptomes, editomes and candidate RNA editing factors in the hornwort Anthoceros agrestis

   https://doi.org/10.1111/nph.16297  

   Gerke, Philipp ; Szövényi, Péter ; Neubauer, Anna ; Lenz, Henning ; Gutmann, Bernard ; McDowell, Rose ; Small, Ian ; Schallenberg‐Rüdinger, Mareike ; Knoop, Volker ( December 2019 , New Phytologist)

   Hornworts are crucial to understand the phylogeny of early land plants. The emergence of ‘reverse’ U‐to‐C RNA editing accompanying the widespread C‐to‐U RNA editing in plant chloroplasts and mitochondria may be a molecular synapomorphy of a hornwort–tracheophyte clade. C‐to‐U RNA editing is well understood after identification of many editing factors in models likeArabidopsis thalianaandPhyscomitrella patens, but there is no plant model yet to investigate U‐to‐C RNA editing. The hornwortAnthoceros agrestisis now emerging as such a model system.

   We report on the assembly and analyses of theA. agrestischloroplast and mitochondrial genomes, their transcriptomes and editomes, and a large nuclear gene family encoding pentatricopeptide repeat (PPR) proteins likely acting as RNA editing factors.

   Both organelles inA. agrestisfeature high amounts of RNA editing, with altogether > 1100 sites of C‐to‐U and 1300 sites of U‐to‐C editing. The nuclear genome reveals > 1400 genes for PPR proteins with variable carboxyterminal DYW domains.

   We observe significant variants of the ‘classic’ DYW domain, in the meantime confirmed as the cytidine deaminase for C‐to‐U editing, and discuss the first attractive candidates for reverse editing factors given their excellent matches to U‐to‐C editing targets according to the PPR‐RNA binding code.

    

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2. RNA-editing enzymes ADAR1 and ADAR2 coordinately regulate the editing and expression of Ctn RNA

   https://doi.org/10.1002/1873-3468.12795  

   Anantharaman, Aparna ; Gholamalamdari, Omid ; Khan, Abid ; Yoon, Je-Hyun ; Jantsch, Michael F. ; Hartner, Jochen C. ; Gorospe, Myriam ; Prasanth, Supriya G. ; Prasanth, Kannanganattu V. ( September 2017 , FEBS Letters)
3. CRISPR‐Cas9 Genome Editing in the Moss Physcomitrium (Formerly Physcomitrella ) patens

   https://doi.org/10.1002/cpz1.725  

   Wu, Shu‐Zon ; Ryken, Samantha E. ; Bezanilla, Magdalena ( April 2023 , Current Protocols)

   Until recently, precise genome editing has been limited to a few organisms. The ability of Cas9 to generate double stranded DNA breaks at specific genomic sites has greatly expanded molecular toolkits in many organisms and cell types. Before CRISPR‐Cas9 mediated genome editing,P. patenswas unique among plants in its ability to integrate DNA via homologous recombination. However, selection for homologous recombination events was required to obtain edited plants, limiting the types of editing that were possible. Now with CRISPR‐Cas9, molecular manipulations inP. patenshave greatly expanded. This protocol describes a method to generate a variety of different genome edits. The protocol describes a streamlined method to generate the Cas9/sgRNA expression constructs, design homology templates, transform, and quickly genotype plants. © 2023 Wiley Periodicals LLC.

   Basic Protocol 1: Constructing the Cas9/sgRNA transient expression vector

   Alternate Protocol 1: Shortcut to generating single and pooled Cas9/sgRNA expression vectors

   Basic Protocol 2: Designing the oligonucleotide‐based homology‐directed repair (HDR) template

   Alternate Protocol 2: Designing the plasmid‐based HDR template

   Basic Protocol 3: Inducing genome editing by transforming CRISPR vector intoP. patensprotoplasts

   Basic Protocol 4: Identifying edited plants.

    

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4. Identification of rfk-1 , a Meiotic Driver Undergoing RNA Editing in Neurospora

   https://doi.org/10.1534/genetics.119.302122  

   Rhoades, Nicholas A. ; Harvey, Austin M. ; Samarajeewa, Dilini A. ; Svedberg, Jesper ; Yusifov, Aykhan ; Abusharekh, Anna ; Manitchotpisit, Pennapa ; Brown, Daren W. ; Sharp, Kevin J. ; Rehard, David G. ; et al ( May 2019 , Genetics)
5. Integrated Genome and Protein Editing Swaps α ‐2,6 Sialylation for α ‐2,3 Sialic Acid on Recombinant Antibodies from CHO

   https://doi.org/10.1002/biot.201600502  

   Chung, Cheng‐yu ; Wang, Qiong ; Yang, Shuang ; Yin, Bojiao ; Zhang, Hui ; Betenbaugh, Michael ( January 2017 , Biotechnology Journal)

   Immunoglobin G withα‐2,6 sialylation has been reported to have an impact on antibody‐dependent cellular cytotoxicity and anti‐inflammatory efficacy. However, production of antibodies withα‐2,6 sialylation from Chinese hamster ovary cells is challenging due to the inaccessibility of sialyltransferases for the heavy chain N‐glycan site and the presence of exclusivelyα‐2,3 sialyltransferases. In this study, combining mutations on the Fc regions to allow sialyltransferase accessibility with overexpression ofα‐2,6 sialyltransferase produced IgG with significant levels of bothα‐2,6 andα‐2,3 sialylation. Therefore, ST3GAL4 and ST3GAL6 genes were disrupted by CRISPR/Cas9 to minimize theα‐2,3 sialylation. Sialidase treatment and SNA lectin blot indicated greatly increasedα‐2,6 sialylation level relative toα‐2,3 sialylation for theα‐2,3 sialyltransferase knockouts when combined withα‐2,6 sialyltransferase overexpression. Indeed,α‐2,3 linked sialic acids were not detected on IgG produced from theα‐2,3 sialyltransferase knockout‐α‐2,6 sialyltransferase overexpression pools. Finally, glycoprofiling of IgG with four amino acid substitutions expressed from anα‐2,3 sialyltransferase knockout‐α‐2,6 sialyltransferase stable clone resulted in more than 77% sialylated glycans and more than 62% biantennary disialylated glycans as indicated by both MALDI‐TOF and LC‐ESI‐MS. Engineered antibodies from these modified Chinese hamster ovary cell lines will provide biotechnologists with IgGs containing N‐glycans with different structural variations for examining the role of glycosylation on protein performance.

    

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