Journal of Genetics and Genomics

TALENs: Customizable Molecular DNA Scissors for Genome Engineering of Plants

Kunling Chen, Caixia Gao

2013, 40(6): 271-279. doi: 10.1016/j.jgg.2013.03.009

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Abstract:
Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology. Transcription activator-like effector nucleases (TALENs), consisting of an engineered specific (TALE) DNA binding domain and a Fok I cleavage domain, are newly developed versatile reagents for genome engineering in different organisms. Because of the simplicity of the DNA recognition code and their modular assembly, TALENs can act as customizable molecular DNA scissors inducing double-strand breaks (DSBs) at given genomic location. Thus, they provide a valuable approach to targeted genome modifications such as mutations, insertions, replacements or chromosome rearrangements. In this article, we review the development of TALENs, and summarize the principles and tools for TALEN-mediated gene targeting in plant cells, as well as current and potential strategies for use in plant research and crop improvement.

TALEN or Cas9 – Rapid, Efficient and Specific Choices for Genome Modifications

Chuanxian Wei, Jiyong Liu, Zhongsheng Yu, Bo Zhang, Guanjun Gao, Renjie Jiao

2013, 40(6): 281-289. doi: 10.1016/j.jgg.2013.03.013

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Abstract:
Precise modifications of complex genomes at the single nucleotide level have been one of the big goals for scientists working in basic and applied genetics, including biotechnology, drug development, gene therapy and synthetic biology. However, the relevant techniques for making these manipulations in model organisms and human cells have been lagging behind the rapid high throughput studies in the post-genomic era with a bottleneck of low efficiency, time consuming and laborious manipulation, and off-targeting problems. Recent discoveries of TALEs (transcription activator-like effectors) coding system and CRISPR (clusters of regularly interspaced short palindromic repeats) immune system in bacteria have enabled the development of customized TALENs (transcription activator-like effector nucleases) and CRISPR/Cas9 to rapidly edit genomic DNA in a variety of cell types, including human cells, and different model organisms at a very high efficiency and specificity. In this review, we first briefly summarize the development and applications of TALENs and CRISPR/Cas9-mediated genome editing technologies; compare the advantages and constraints of each method; particularly, discuss the expected applications of both techniques in the field of site-specific genome modification and stem cell based gene therapy; finally, propose the future directions and perspectives for readers to make the choices.

Short Tandem Target Mimic: A Long Journey to the Engineered Molecular Landmine for Selective Destruction/Blockage of MicroRNAs in Plants and Animals

Guiliang Tang, Xiaoqing Tang

2013, 40(6): 291-296. doi: 10.1016/j.jgg.2013.02.004

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MicroRNAs (miRNAs) are a population of highly conserved specific small ribo-regulators that negatively regulate gene expressions in both plants and animals. They play a key role in post-transcriptional gene regulation by destabilizing the target gene transcripts or blocking protein translation from them. Interestingly, these negative regulators are largely compromised by an upstream layer of negative regulators “target mimics” found in plants or “endogenous competing RNAs” revealed recently in animals. These endogenous regulatory mechanisms of “double negatives making a positive” have now been developed into a key strategy in the study of small RNA functions. This review presents some reflections on the long journey to the short tandem target mimic (STTM) for selective destruction/blockage of specific miRNAs in plants and animals, and the potential applications of STTM are discussed.

Characterization of the Drosophila Atlastin Interactome Reveals VCP as a Functionally Related Interactor

Niamh C. O'Sullivan, Nina Dräger, Cahir J. O'Kane

2013, 40(6): 297-306. doi: 10.1016/j.jgg.2013.04.008

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Abstract:
At least 25 genes, many involved in trafficking, localisation or shaping of membrane organelles, have been identified as causative genes for the neurodegenerative disorder hereditary spastic paraplegia (HSP). One of the most commonly mutated HSP genes, atlastin-1, encodes a dynamin-like GTPase that mediates homotypic fusion of endoplasmic reticulum (ER) membranes. However, the molecular mechanisms of atlastin-1-related membrane fusion and axonopathy remain unclear. To better understand its mode of action, we used affinity purification coupled with mass spectrometry to identify protein interactors of atlastin in Drosophila. Analysis of 72 identified proteins revealed that the atlastin interactome contains many proteins involved in protein processing and transport, in addition to proteins with roles in mRNA binding, metabolism and mitochondrial proteins. The highest confidence interactor from mass spectrometry analysis, the ubiquitin-selective AAA-ATPase valosin-containing protein (VCP), was validated as an atlastin-interacting protein, and VCP and atlastin showed overlapping subcellular distributions. Furthermore, VCP acted as a genetic modifier of atlastin: loss of VCP partially suppressed an eye phenotype caused by atlastin overexpression, whereas overexpression of VCP enhanced this phenotype. These interactions between atlastin and VCP suggest a functional relationship between these two proteins, and point to potential shared mechanisms between HSP and other forms of neurodegeneration.

Characterization and Fine Mapping of a Necrotic Leaf Mutant in Maize (Zea mays L.)

Lijing Wang, Shuai Han, Shiyi Zhong, Haizhong Wei, Yanjun Zhang, Yan Zhao, Baoshen Liu

2013, 40(6): 307-314. doi: 10.1016/j.jgg.2013.04.004

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Maize (Zea mays L.) is a commercially important crop. Its yield can be reduced by mutations in biosynthetic and degradative pathways that cause death. In this paper, we describe the necrotic leaf (nec-t) mutant, which was obtained from an inbred line, 81647. The nec-t mutant plants had yellow leaves with necrotic spots, reduced chlorophyll content, and the etiolated seedlings died under normal growth conditions. Transmission electron microscopy revealed scattered thylakoids, and reduced numbers of grana lamellae and chloroplasts per cell. Histochemical staining suggested that spot formation of nec-t leaves might be due to cell death. Genetic analysis showed that necrosis was caused by the mutation of a recessive locus. Using simple sequence repeat markers, the Nec-t gene was mapped between mmc0111 and bnlg2277 on the short arm of chromosome 2. A total of 1287 individuals with the mutant phenotype from a F₂ population were used for physical mapping. The Nec-t gene was located between markers T31 and H8 within a physical region of 131.7 kb.

Lack of an Additive Effect between the Deletions of Klf5 and Nkx3-1 in Mouse Prostatic Tumorigenesis

Changsheng Xing, Xiaoying Fu, Xiaodong Sun, Jin-Tang Dong

2013, 40(6): 315-318. doi: 10.1016/j.jgg.2013.04.005

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Directed Evolution of Insoluble Arabidopsis thaliana Zeta Class Glutathione S-Transferase Mutants for Higher Solubility in Escherichia coli

Yicun Wang, Feng Zhang, Haiwei Chen, Xiwen Chen, Defu Chen

2013, 40(6): 319-322. doi: 10.1016/j.jgg.2013.03.005

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2013 Vol. 40, No. 6