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1. High‐efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR /Cas9

   https://doi.org/10.1111/tpj.13446  

   Gil‐Humanes, Javier ; Wang, Yanpeng ; Liang, Zhen ; Shan, Qiwei ; Ozuna, Carmen V. ; Sánchez‐León, Susana ; Baltes, Nicholas J. ; Starker, Colby ; Barro, Francisco ; Gao, Caixia ; et al ( February 2017 , The Plant Journal)

   The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence‐specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved usingAgrobacteriumT‐DNA, biolistics or by stably integrating nuclease‐encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such asNicotinana tabacum(tobacco) andSolanum lycopersicum(tomato), greater than 10‐fold enhancements inGTfrequencies have been achieved usingDNAvirus‐based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundantSSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon‐based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110‐fold increase in expression of a reporter gene relative to non‐replicating controls. Furthermore, replicons carryingCRISPR/Cas9 nucleases and repair templates achievedGTat an endogenousubiquitinlocus at frequencies 12‐fold greater than non‐viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these highGTfrequencies. We also demonstrate gene‐targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with theWDVreplicons, multiplexedGTwithin the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies ofGTusingWDV‐basedDNAreplicons will make it possible to edit complex cereal genomes without the need to integrateGTreagents into the genome.

    

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2. CRISPR–Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells

   https://doi.org/10.1093/nar/gkac049  

   Lin-Shiao, Enrique ; Pfeifer, Wolfgang G. ; Shy, Brian R. ; Saffari Doost, Mohammad ; Chen, Evelyn ; Vykunta, Vivasvan S. ; Hamilton, Jennifer R. ; Stahl, Elizabeth C. ; Lopez, Diana M. ; Sandoval Espinoza, Cindy R. ; et al ( February 2022 , Nucleic Acids Research)

   DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR–Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei.

    

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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. Accurate measurement of transgene copy number in crop plants using droplet digital PCR

   https://doi.org/10.1111/tpj.13517  

   Collier, Ray ; Dasgupta, Kasturi ; Xing, Yan‐Ping ; Hernandez, Bryan Tarape ; Shao, Min ; Rohozinski, Dominica ; Kovak, Emma ; Lin, Jeanie ; de Oliveira, Maria Luiza P. ; Stover, Ed ; et al ( April 2017 , The Plant Journal)

   Genetic transformation is a powerful means for the improvement of crop plants, but requires labor‐ and resource‐intensive methods. An efficient method for identifying single‐copy transgene insertion events from a population of independent transgenic lines is desirable. Currently, transgene copy number is estimated by either Southern blot hybridization analyses or quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization is a convincing and reliable method, but it also is expensive, time‐consuming and often requires a large amount of genomicDNAand radioactively labeled probes. Alternatively,qPCRrequires lessDNAand is potentially simpler to perform, but its results can lack the accuracy and precision needed to confidently distinguish between one‐ and two‐copy events in transgenic plants with large genomes. To address this need, we developed a droplet digitalPCR‐based method for transgene copy number measurement in an array of crops: rice, citrus, potato, maize, tomato and wheat. The method utilizes specific primers to amplify target transgenes, and endogenous reference genes in a single duplexed reaction containing thousands of droplets. Endpoint amplicon production in the droplets is detected and quantified using sequence‐specific fluorescently labeled probes. The results demonstrate that this approach can generate confident copy number measurements in independent transgenic lines in these crop species. This method and the compendium of probes and primers will be a useful resource for the plant research community, enabling the simple and accurate determination of transgene copy number in these six important crop species.

    

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5. Na + /K + ‐ ATP ase gene duplications in clitellate annelids are associated with freshwater colonization

   https://doi.org/10.1111/jeb.13439  

   Horn, Kevin M. ; Williams, Bronwyn W. ; Erséus, Christer ; Halanych, Kenneth M. ; Santos, Scott R. ; Creuzé des Châtelliers, Michel ; Anderson, Frank E. ( April 2019 , Journal of Evolutionary Biology)

   Major habitat transitions, such as those from marine to freshwater habitats or from aquatic to terrestrial habitats, have occurred infrequently in animal evolution and may represent a barrier to diversification. Identifying genomic events associated with these transitions can help us better understand mechanisms that allow animals to cross these barriers and diversify in new habitats. Study of theCapitella telataandHelobdella robustagenomes allows examination of one such habitat transition (marine to freshwater) in Annelida. Initial examination of these genomes indicated that the freshwater leechH. robustacontains many more copies (12) of the sodium–potassium pump alpha‐subunit (Na⁺/K⁺‐ATPase) gene than does the marine polychaeteC. telata(2). The sodium–potassium pump plays a key role in maintenance of cellular ionic balance and osmoregulation, and Na⁺/K⁺‐ATPase duplications may have helped annelids invade and diversify in freshwater habitats. To assess whether the timing of Na⁺/K⁺‐ATPase duplications coincided with the marine‐to‐freshwater transition in Clitellata, we used transcriptomic data from 18 annelid taxa, along with the two genomes, to infer a species phylogeny and identified Na⁺/K⁺‐ATPase gene transcripts in order to infer the timing of gene duplication events using tree‐based methods. The inferred timing of Na⁺/K⁺‐ATPase duplication events is consistent with the timing of the initial marine‐to‐freshwater transition early in the history of clitellate annelids, supporting the hypothesis that gene duplications may have played a role in the annelid diversification into freshwater habitats.

    

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