scholarly journals Targeted mutagenesis of the SUMO protease, Overly Tolerant to Salt1 in rice through CRISPR/Cas9-mediated genome editing reveals a major role of this SUMO protease in salt tolerance

2019 ◽  
Author(s):  
Cunjin Zhang ◽  
Anjil Kumar Srivastava ◽  
Ari Sadanandom
Keyword(s):  
Gene Family ◽  
Genome Editing ◽  
Gene Editing ◽  
Family Members ◽  
Targeted Mutagenesis ◽  
Specific Gene ◽  
Unexpected Finding ◽  
Sumo Protease ◽  
Sumo Proteases

SUMO proteases are encoded by a large gene family in rice and are a potential source of specificity within the SUMO system that is responding to different environmental cues. We previously demonstrated a critical role of OsOTS class of SUMO proteases in salt and drought stress in rice by silencing several family members collectively via RNAi methods. However, to date it has not been possible to assign a role to specific family members due to limitations of RNAi mediated off target silencing across several members of the gene family. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease 9 (CRISPR/Cas9) system has emerged as a promising technology for specific gene editing in crop plants. Here, we demonstrate targeted mutagenesis of OsOTS1 in rice using the CRISPR/Cas9 gene editing system in the rice cultivar Kitaake. Guide RNA mediated mutations in OsOTS1 was highly efficient with almost 95% of T0 transgenics showing the desired effect with no off-target mutations. The OsOTS1 mutations observed in T0 generation were heritable in subsequent generations. OsOTS1 CRISPR lines show enhanced sensitivity to salt with reduced root and shoot biomass indicating that OsOTS1 has a major role in salt stress tolerance in rice. This unexpected finding indicates that precise and effective genome editing can be used to characterise specificity within the SUMO system in rice.

scholarly journals Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuu Asano ◽  
Kensuke Yamashita ◽  
Aoi Hasegawa ◽  
Takanori Ogasawara ◽  
Hoshie Iriki ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dictyostelium Discoideum ◽  
Streptococcus Pyogenes ◽  
Nucleotide Substitution ◽  
Point Mutations ◽  
Targeted Mutagenesis ◽  
Single Amino Acid ◽  
Specific Gene ◽  
Knock Out ◽  
Protospacer Adjacent Motif

AbstractThe powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

Role of CCN, a vertebrate specific gene family, in development

2008 ◽  
Vol 51 (1) ◽  
pp. 55-67 ◽  
Author(s):  
Ken-ichi Katsube ◽  
Kei Sakamoto ◽  
Yoshihiro Tamamura ◽  
Akira Yamaguchi
Keyword(s):  
Gene Family ◽  
Specific Gene

scholarly journals Essential Role of Nuclear Localization for Yeast Ulp2 SUMO Protease Function

2009 ◽  
Vol 20 (8) ◽  
pp. 2196-2206 ◽  
Author(s):  
Mary B. Kroetz ◽  
Dan Su ◽  
Mark Hochstrasser
Keyword(s):  
Nuclear Localization ◽  
Nuclear Protein ◽  
Catalytic Domain ◽  
Yeast Growth ◽  
Nuclear Targeting ◽  
Sumo Protease ◽  
Sumo Proteases ◽  
Localization Signal

The SUMO protein is covalently attached to many different substrates throughout the cell. This modification is rapidly reversed by SUMO proteases. The Saccharomyces cerevisiae SUMO protease Ulp2 is a nuclear protein required for chromosome stability and cell cycle restart after checkpoint arrest. Ulp2 is related to the human SENP6 protease, also a nuclear protein. All members of the Ulp2/SENP6 family of SUMO proteases have large but poorly conserved N-terminal domains (NTDs) adjacent to the catalytic domain. Ulp2 also has a long C-terminal domain (CTD). We show that CTD deletion has modest effects on yeast growth, but poly-SUMO conjugates accumulate. In contrast, the NTD is essential for Ulp2 function and is required for nuclear targeting. Two short, widely separated sequences within the NTD confer nuclear localization. Efficient Ulp2 import into the nucleus requires the β-importin Kap95, which functions on classical nuclear-localization signal (NLS)-bearing substrates. Remarkably, replacement of the entire >400-residue NTD by a heterologous NLS results in near-normal Ulp2 function. These data demonstrate that nuclear localization of Ulp2 is crucial in vivo, yet only small segments of the NTD provide the key functional elements, explaining the minimal sequence conservation of the NTDs in the Ulp2/SENP6 family of enzymes.

scholarly journals The Three Clades of the Telomere-AssociatedTLOGene Family of Candida albicans Have Different Splicing, Localization, and Expression Features

2012 ◽  
Vol 11 (10) ◽  
pp. 1268-1275 ◽  
Author(s):  
Matthew Z. Anderson ◽  
Joshua A. Baller ◽  
Keely Dulmage ◽  
Lauren Wigen ◽  
Judith Berman
Keyword(s):  
Candida Albicans ◽  
Gene Family ◽  
Transcription Regulation ◽  
Family Members ◽  
Gene Products ◽  
Content Type ◽  
Human Host ◽  
Wide Range ◽  
N Terminus

ABSTRACTCandida albicansgrows within a wide range of host niches, and this adaptability enhances its success as a commensal and as a pathogen. The telomere-associatedTLOgene family underwent a recent expansion from one or two copies in other CUG clade members to 14 expressed copies inC. albicans. This correlates with increased virulence and clinical prevalence relative to those of otherCandidaclade species. The 14 expressedTLOgene family members have a conserved Med2 domain at the N terminus, suggesting a role in general transcription. The C-terminal half is more divergent, distinguishing three clades: clade α and clade β have no introns and encode proteins that localize primarily to the nucleus; clade γ sometimes undergoes splicing, and the gene products localize within the mitochondria as well as the nuclei. Additionally,TLOα genes are generally expressed at much higher levels than areTLOγ genes. We propose that expansion of theTLOgene family and the predicted role of Tlo proteins in transcription regulation provideC. albicanswith the ability to adapt rapidly to the broad range of different environmental niches within the human host.

scholarly journals Gene editing in dermatology: Harnessing CRISPR for the treatment of cutaneous disease

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 281
Author(s):  
Catherine Baker ◽  
Matthew S. Hayden
Keyword(s):  
Human Genome ◽  
Genome Editing ◽  
Gene Editing ◽  
Human Diseases ◽  
Therapeutic Application ◽  
Cutaneous Disease ◽  
Crispr System ◽  
Novel Approaches

The discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system has revolutionized gene editing research. Through the repurposing of programmable RNA-guided CRISPR-associated (Cas) nucleases, CRISPR-based genome editing systems allow for the precise modification of specific sites in the human genome and inspire novel approaches for the study and treatment of inherited and acquired human diseases. Here, we review how CRISPR technologies have stimulated key advances in dermatologic research.  We discuss the role of CRISPR in genome editing for cutaneous disease and highlight studies on the use of CRISPR-Cas technologies for genodermatoses, cutaneous viruses and bacteria, and melanoma. Additionally, we examine key limitations of current CRISPR technologies, including the challenges these limitations pose for the widespread therapeutic application of CRISPR-based therapeutics.

scholarly journals Genome-wide identification and expression profiling of invertase gene family for abiotic stresses tolerance in Poncirus trifoliata

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Bachar Dahro ◽  
Yue Wang ◽  
Ahmed Alhag ◽  
Chunlong Li ◽  
Dayong Guo ◽  
...  
Keyword(s):  
Gene Family ◽  
Abiotic Stresses ◽  
Family Members ◽  
Physiological Role ◽  
Poncirus Trifoliata ◽  
High Accumulation ◽  
Functional Studies ◽  
Genome Wide ◽  
Cold Tolerant

Abstract Background Sucrose (Suc) hydrolysis is directly associated with plants tolerance to multiple abiotic stresses. Invertase (INV) enzymes irreversibly catalyze Suc degradation to produce glucose (Glc) and fructose (Frc). However, genome-wide identification and function of individual members of the INV gene family in Poncirus trifoliata or its Citrus relatives in response to abiotic stresses are not fully understood. Results In this report, fourteen non-redundant PtrINV family members were identified in P. trifoliata including seven alkaline/neutral INV genes (PtrA/NINV1–7), two vacuolar INV genes (PtrVINV1–2), and five cell wall INV isoforms (PtrCWINV1–5). A comprehensive analysis based on the biochemical characteristics, the chromosomal location, the exon–intron structures and the evolutionary relationships demonstrated the conservation and the divergence of PtrINVs. In addition, expression analysis of INV genes during several abiotic stresses in various tissues indicated the central role of A/NINV7 among INV family members in response to abiotic stresses. Furthermore, our data demonstrated that high accumulation of Suc, Glc, Frc and total sugar contents were directly correlated with the elevated activities of soluble INV enzymes in the cold-tolerant P. trifoliata, C. ichangensis and C. sinensis, demonstrating the potential role of soluble INV enzymes for the cold tolerance of Citrus. Conclusions This work offered a framework for understanding the physiological role of INV genes and laid a foundation for future functional studies of these genes in response to abiotic stresses.

scholarly journals An updated census of the maize TIFY family

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247271
Author(s):  
Pingdong Sun ◽  
Yannan Shi ◽  
Aga Guido Okwana Valerio ◽  
Eli James Borrego ◽  
Qingyun Luo ◽  
...  
Keyword(s):  
Gene Family ◽  
Expression Analysis ◽  
Family Members ◽  
Specific Gene ◽  
Ecological Analysis ◽  
Crop Species ◽  
Conserved Domains ◽  
Important Crop ◽  
Ear Motif ◽  
Domain Protein

The TIFY gene family is a plant-specific gene family encoding a group of proteins characterized by its namesake, the conservative TIFY domain and members can be organized into four subfamilies: ZML, TIFY, PPD and JAZ (Jasmonate ZIM-domain protein) by presence of additional conserved domains. The TIFY gene family is intensively explored in several model and agriculturally important crop species and here, yet the composition of the TIFY family of maize has remained unresolved. This study increases the number of maize TIFY family members known by 40%, bringing the total to 47 including 38 JAZ, 5 TIFY, and 4 ZML genes. The majority of the newly identified genes were belonging to the JAZ subfamily, six of which had aberrant TIFY domains, suggesting loss JAZ-JAZ or JAZ-NINJA interactions. Six JAZ genes were found to have truncated Jas domain or an altered degron motif, suggesting resistance to classical JAZ degradation. In addition, seven membranes were found to have an LxLxL-type EAR motif which allows them to recruit TPL/TPP co-repressors directly without association to NINJA. Expression analysis revealed that ZmJAZ14 was specifically expressed in the seeds and ZmJAZ19 and 22 in the anthers, while the majority of other ZmJAZs were generally highly expressed across diverse tissue types. Additionally, ZmJAZ genes were highly responsive to wounding and JA treatment. This study provides a comprehensive update of the maize TIFY/JAZ gene family paving the way for functional, physiological, and ecological analysis.

scholarly journals Role of Murine Cytomegalovirus US22 Gene Family Members in Replication in Macrophages

2003 ◽  
Vol 77 (10) ◽  
pp. 5557-5570 ◽  
Author(s):  
Carine Ménard ◽  
Markus Wagner ◽  
Zsolt Ruzsics ◽  
Karina Holak ◽  
Wolfram Brune ◽  
...  
Keyword(s):  
Gene Family ◽  
Family Members ◽  
Cell Types ◽  
Murine Cytomegalovirus ◽  
Sequence Motifs ◽  
Gene Deletions ◽  
Conserved Sequence ◽  
Growth Properties ◽  
Growth Phenotype

ABSTRACT The large cytomegalovirus (CMV) US22 gene family, found in all betaherpesviruses, comprises 12 members in both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV). Conserved sequence motifs suggested a common ancestry and related functions for these gene products. Two members of this family, m140 and m141, were recently shown to affect MCMV replication on macrophages. To test the role of all US22 members in cell tropism, we analyzed the growth properties in different cell types of MCMV mutants carrying transposon insertions in all 12 US22 gene family members. When necessary, additional targeted mutants with gene deletions, ATG deletions, and ectopic gene revertants were constructed. Mutants with disruption of genes M23, M24, m25.1, m25.2, and m128 (ie2) showed no obvious growth phenotype, whereas growth of M43 mutants was reduced in a number of cell lines. Genes m142 and m143 were shown to be essential for virus replication. Growth of mutants with insertions into genes M36, m139, m140, and m141 in macrophages was severely affected. The common phenotype of the m139, m140, and m141 mutants was explained by an interaction at the protein level. The M36-dependent macrophage growth phenotype could be explained by the antiapoptotic function of the gene that was required for growth on macrophages but not for growth on other cell types. Together, the comprehensive set of mutants of the US22 gene family suggests that individual family members have diverged through evolution to serve a variety of functions for the virus.

scholarly journals ROLE OF CRISPR TO IMPROVE ABIOTIC STRESS TOLERANCE IN CROP PLANTS

2021 ◽  
Vol 2021 (1) ◽  
Author(s):  
MU Farooq ◽  
MF Bashir ◽  
MUS Khan ◽  
B Iqbal ◽  
Q Ali
Keyword(s):  
Abiotic Stress ◽  
Genome Editing ◽  
Gene Editing ◽  
Abiotic Stress Tolerance ◽  
Genetically Engineered ◽  
Transcription Activator ◽  
Guide Rnas ◽  
Field Crops ◽  
Genetically Engineered Crops

The study for genetic variation in plant genomes for a variety of crops, as well as developments of genome editing techniques, have made it possible to cultivate for about any desired trait. Zinc finger enzymes; have made strides in genome-editing. Molecular biologists can now more specifically target every gene using transcription activator-like effector nucleases and ZFNs. These methods, on the other hand, are expensive and time-consuming because they involve complex procedures. Referring to various genome editing techniques, CRISPR/Cas9 genetic modification is simple to construct and clone and the Cas9 could be used with different guide RNAs controlling different genes. Following solid evidence demonstrations using the main CRISPR-Cas9 unit in field crops, multiple updated Cas9 cassettes are often used in plant species to improve target precision and reduce off target cleavage. Nmcas9, Sacas9, as well as Stcas9 are a few examples. Furthermore, Cas9 enzymes are readily available from a variety of sources. Bacteria that had never been discovered before has found solutions available to improve specificity and efficacy of gene editing techniques. The choices are summarized in this analysis to plant's experiment to develop crops using CRISPR/Cas9 technology; the tolerance of biotic & abiotic stress may be improved. These strategies will lead to the growth of non-genetically engineered crops with the target phenotype, which will further improve yield capacity under biotic & abiotic stress environments.

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