scholarly journals Generating and characterizing single- and multigene mutants of the Rubisco small subunit family in Arabidopsis

2020 ◽  
Vol 71 (19) ◽  
pp. 5963-5975 ◽  
Author(s):  
Panupon Khumsupan ◽  
Marta A Kozlowska ◽  
Douglas J Orr ◽  
Andreas I Andreou ◽  
Naomi Nakayama ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Catalytic Efficiency ◽  
Small Subunit ◽  
Multiple Gene ◽  
Functional Roles ◽  
Knockout Mutants ◽  
Dna Insertion ◽  
Low Levels ◽  
Rbcs Genes ◽  
Viable Approach

Abstract The primary CO2-fixing enzyme Rubisco limits the productivity of plants. The small subunit of Rubisco (SSU) can influence overall Rubisco levels and catalytic efficiency, and is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. However, SSUs are encoded by a family of nuclear rbcS genes in plants, which makes them challenging to engineer and study. Here we have used CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] and T-DNA insertion lines to generate a suite of single and multiple gene knockout mutants for the four members of the rbcS family in Arabidopsis, including two novel mutants 2b3b and 1a2b3b. 1a2b3b contained very low levels of Rubisco (~3% relative to the wild-type) and is the first example of a mutant with a homogenous Rubisco pool consisting of a single SSU isoform (1B). Growth under near-outdoor levels of light demonstrated Rubisco-limited growth phenotypes for several SSU mutants and the importance of the 1A and 3B isoforms. We also identified 1a1b as a likely lethal mutation, suggesting a key contributory role for the least expressed 1B isoform during early development. The successful use of CRISPR/Cas here suggests that this is a viable approach for exploring the functional roles of SSU isoforms in plants.

scholarly journals Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

Archaea ◽  
2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Shoji Suzuki ◽  
Norio Kurosawa
Keyword(s):  
Gene Knockout ◽  
Arginine Decarboxylase ◽  
Sulfolobus Acidocaldarius ◽  
Dna Photolyase ◽  
Genetic Studies ◽  
Multiple Gene ◽  
Plasmid Construction ◽  
Pcr Product ◽  
Optimized Protocol ◽  
One Step

Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/μg DNA) by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER). In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr-) and arginine decarboxylase- (argD-) deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.

scholarly journals Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

2019 ◽  
Author(s):  
Gabriel A. Suárez ◽  
Kyle R. Dugan ◽  
Brian A. Renda ◽  
Sean P. Leonard ◽  
Lakshmi S. Gangavarapu ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Single Gene ◽  
Golden Gate ◽  
Acinetobacter Baylyi ◽  
Gene Deletions ◽  
Multiple Gene ◽  
A Genome ◽  
Strain Collection ◽  
Acinetobacter Baylyi Adp1 ◽  
Improved Methods

ABSTRACTOne goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 19 successful multiple-gene deletions ranged in size from 21 to 183 kilobases and collectively accounted for 24.6% of its genome. Deletion success could only be partially predicted on the basis of a single-gene knockout strain collection and a new Tn-Seq experiment. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

scholarly journals Specific interaction of an RNA-binding protein with the 3′-UTR of its target mRNA is critical to oomycete sexual reproduction

2021 ◽  
Vol 17 (10) ◽  
pp. e1010001
Author(s):  
Hui Feng ◽  
Chuanxu Wan ◽  
Zhichao Zhang ◽  
Han Chen ◽  
Zhipeng Li ◽  
...  
Keyword(s):  
Sexual Reproduction ◽  
Rna Binding ◽  
Gene Knockout ◽  
Binding Protein ◽  
Mrna Level ◽  
Specific Interaction ◽  
Rna Binding Protein ◽  
Pythium Ultimum ◽  
Knockout Mutants ◽  
Oospore Formation

Sexual reproduction is an essential stage of the oomycete life cycle. However, the functions of critical regulators in this biological process remain unclear due to a lack of genome editing technologies and functional genomic studies in oomycetes. The notorious oomycete pathogen Pythium ultimum is responsible for a variety of diseases in a broad range of plant species. In this study, we revealed the mechanism through which PuM90, a stage-specific Puf family RNA-binding protein, regulates oospore formation in P. ultimum. We developed the first CRISPR/Cas9 system-mediated gene knockout and in situ complementation methods for Pythium. PuM90-knockout mutants were significantly defective in oospore formation, with empty oogonia or oospores larger in size with thinner oospore walls compared with the wild type. A tripartite recognition motif (TRM) in the Puf domain of PuM90 could specifically bind to a UGUACAUA motif in the mRNA 3′ untranslated region (UTR) of PuFLP, which encodes a flavodoxin-like protein, and thereby repress PuFLP mRNA level to facilitate oospore formation. Phenotypes similar to PuM90-knockout mutants were observed with overexpression of PuFLP, mutation of key amino acids in the TRM of PuM90, or mutation of the 3′-UTR binding site in PuFLP. The results demonstrated that a specific interaction of the RNA-binding protein PuM90 with the 3′-UTR of PuFLP mRNA at the post-transcriptional regulation level is critical for the sexual reproduction of P. ultimum.

scholarly journals Genetically Altered Mouse Models: the Good, the Bad, and the Ugly

2003 ◽  
Vol 14 (3) ◽  
pp. 154-174 ◽  
Author(s):  
Tamizchelvi Thyagarajan ◽  
Satish Totey ◽  
Mary Jo S. Danton ◽  
Ashok B. Kulkarni
Keyword(s):  
Gene Targeting ◽  
Mouse Models ◽  
Molecular Mechanisms ◽  
Gene Knockout ◽  
Genetically Engineered ◽  
Murine Models ◽  
Functional Roles ◽  
Targeted Gene Disruption ◽  
Gene Knockout Mouse

Targeted gene disruption in mice is a powerful tool for generating murine models for human development and disease. While the human genome program has helped to generate numerous candidate genes, few genes have been characterized for their precise in vivo functions. Gene targeting has had an enormous impact on our ability to delineate the functional roles of these genes. Many gene knockout mouse models faithfully mimic the phenotypes of the human diseases. Because some models display an unexpected or no phenotype, controversy has arisen about the value of gene-targeting strategies. We argue in favor of gene-targeting strategies, provided they are used with caution, particularly in interpreting phenotypes in craniofacial and oral biology, where many genes have pleiotropic roles. The potential pitfalls are outweighed by the unique opportunities for developing and testing different therapeutic strategies before they are introduced into the clinic. In the future, we believe that genetically engineered animal models will be indispensable for gaining important insights into the molecular mechanisms underlying development, as well as disease pathogenesis, diagnosis, prevention, and treatment.

Efficient circular gene knockout system for Burkholderiales strain DSM 7029 and Mycobacterium smegmatis mc2 155

2019 ◽  
Vol 51 (7) ◽  
pp. 697-706 ◽  
Author(s):  
Xiaolai Lei ◽  
Qiuxia Fan ◽  
Tian Huang ◽  
Haiyun Liu ◽  
Guoping Zhao ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Gene Knockout ◽  
Gene Cassette ◽  
Integrase Gene ◽  
Multiple Gene ◽  
System A ◽  
Attachment Sites ◽  
Sacb Gene ◽  
Gene Knockouts ◽  
Serine Integrase

Abstract Multiple gene knockouts are often employed in studies of microbial physiology and genetics. However, the selective markers that confer antibiotic resistance are generally limited, so it is necessary to remove these resistance genes before the next round of using, which is time consuming and labor intensive. Here, we created a universal circular gene knockout system for both the gram-negative bacterial Burkholderiales strain DSM 7029 and the gram-positive bacterial Mycobacterium smegmatis mc2 155, by combining the homologous recombination with multiple serine integrase-meditated site-specific recombination systems. In this system, a resistance gene and an integrase gene were constructed within the two attachment sites corresponding to a second, different integrase to form a cassette for gene disruption, which could be easily removed by the second integrase during the subsequent round of gene knockout. The sacB gene was also employed for negative selection. As the integrase-mediated deletion of the resistance/integrase gene cassette was highly efficient and concurrent with the following knockout round, the cyclic use of three cassettes could achieve multiple gene knockout in a sequential manner. Following the modularity concept in synthetic biology, common components of the knockout plasmids were retained as BioBricks, accelerating the knockout plasmids construction process. The circular gene knockout system can also be used for multiple gene insertions and applied to other microorganisms.

scholarly journals Functional Dissection of an Alternatively Spliced Herpesvirus Gene by Splice Site Mutagenesis

2016 ◽  
Vol 90 (9) ◽  
pp. 4626-4636 ◽  
Author(s):  
Tim Schommartz ◽  
Stefan Loroch ◽  
Malik Alawi ◽  
Adam Grundhoff ◽  
Albert Sickmann ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Viral Replication ◽  
Splice Site ◽  
Gene Knockout ◽  
Single Gene ◽  
Viral Gene ◽  
Gene Products ◽  
Knockout Mutants ◽  
Alternatively Spliced ◽  
Site Mutagenesis

ABSTRACTHerpesviruses have large and complex DNA genomes. The largest among the herpesviruses, those of the cytomegaloviruses, include over 170 genes. Although most herpesvirus gene products are expressed from unspliced transcripts, a substantial number of viral transcripts are spliced. Some viral transcripts are subject to alternative splicing, which leads to the expression of several proteins from a single gene. Functional analysis of individual proteins derived from an alternatively spliced gene is difficult, as deletion and nonsense mutagenesis, both common methods used in the generation of viral gene knockout mutants, affect several or all gene products at the same time. Here, we show that individual gene products of an alternatively spliced herpesvirus gene can be inactivated selectively by mutagenesis of the splice donor or acceptor site and by intron deletion or substitution mutagenesis. We used this strategy to dissect the essential M112/113 gene of murine cytomegalovirus (MCMV), which encodes the MCMV Early 1 (E1) proteins. The expression of each of the four E1 protein isoforms was inactivated individually, and the requirement for each isoform in MCMV replication was analyzed in fibroblasts, endothelial cells, and macrophages. We show that the E1 p87 isoform, but not the p33, p36, and p38 isoforms, is essential for viral replication in cell culture. Moreover, the presence of one of the two medium-size isoforms (p36 or p38) and the presence of intron 1, but not its specific sequence, are required for viral replication. This study demonstrates the usefulness of splice site mutagenesis for the functional analysis of alternatively spliced herpesvirus genes.IMPORTANCEHerpesviruses include up to 170 genes in their DNA genomes. The functions of most viral gene products remain poorly defined. The construction of viral gene knockout mutants has thus been an important tool for functional analysis of viral proteins. However, this strategy is of limited use when viral gene transcripts are alternatively spliced, leading to the expression of several proteins from a single gene. In this study, we showed, as a proof of principle, that each protein product of an alternatively spliced gene can be eliminated individually by splice site mutagenesis. Mutant viruses lacking individual protein products displayed different phenotypes, demonstrating that the products of alternatively spliced genes have nonredundant functions.

Lack of association of transforming growth factor (TGF)-b1 and b2 gene polymorphisms with multiple sclerosis (MS) in Northern Ireland

1999 ◽  
Vol 5 (2) ◽  
pp. 105-109
Author(s):  
G V McDonnell ◽  
C W Kirk ◽  
S A Hawkins ◽  
C A Graham
Keyword(s):  
Transforming Growth Factor ◽  
Gene Knockout ◽  
Association Studies ◽  
B1 Gene ◽  
Relapsing Remitting ◽  
Pcr Products ◽  
Gene Knockout Mice ◽  
Immunosuppressive Cytokine ◽  
Low Levels ◽  
Normal Controls

Objective: To examine the influence of TGF-b genes on MS susceptibility. Background: TGF-b, of which three homologous isoforms exist (1, 2 and 3), is a strongly immunosuppressive cytokine - inhibiting expression of pro-inflammatory cytokines and blocking cytokine induction of adhesion molecules. TGF-b delays onset of EAE and TGF-b1 gene knockout mice develop fatal multifocal inflammatory disease. High TGF-b levels exist during MS remission whilst E-selectin, whose expression is inhibited by TGF-b, is found at higher levels in primary progressive disease (PPMS) and it is postulated that the unremitting course of PPMS may be due to low levels of TGF-b. Methods: Gene association studies using separate polymorphic microsatellite markers for TGF-b1 and TGF-b2 were performed, incorporating 151 relapsing-remitting or secondary progressive MS (RR/SPMS) patients, 104 PPMS patients and 159 normal controls (Nor). Forward primers were 5' end-labelled with 6-Fam, PCR products were analysed on an Applied Biosystems 373A fluorescent fragment analyser and Genescan 672 software was used for allele sizing. Results: No significant differences existed in allele frequencies between either MS group and controls regarding the TGF-b1 marker: RR/SPMS vs Nor (P=0.48, df=8); PPMS vs Nor (P=0.34, df=8). Similarly there were no associations demonstrated with the TGF-b2 marker: RR/SPMS vs Nor (P=0.24, df=2); PPMS vs Nor (P=0.53, df=2). Conclusion: These data indicate that TGF-b1 and b2 genes are not loci influencing MS susceptibility, either RR/SPMS or PPMS, in this population.

scholarly journals The Interleukin-17 Gene of Herpesvirus Saimiri

1998 ◽  
Vol 72 (7) ◽  
pp. 5797-5801 ◽  
Author(s):  
Andrea Knappe ◽  
Christian Hiller ◽  
Henk Niphuis ◽  
François Fossiez ◽  
Mathias Thurau ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Transformation ◽  
Cell Lymphoma ◽  
Gene Knockout ◽  
Herpesvirus Saimiri ◽  
Interleukin 17 ◽  
Wild Type ◽  
Knockout Mutants ◽  
Cottontop Tamarins

ABSTRACT In comparison to wild-type herpesvirus saimiri, viral interleukin-17 gene knockout mutants have unaltered behavior regarding viral replication, T-cell transformation in vitro, and pathogenicity in cottontop tamarins. Thus, this gene is not required for T-cell lymphoma induction but may contribute to apathogenic viral persistence in the natural host, the squirrel monkey.

scholarly journals ASH2L drives proliferation and sensitivity to bleomycin and other genotoxins in Hodgkin’s lymphoma and testicular cancer cells

2020 ◽  
Author(s):  
Daniel Constantin ◽  
Christian Widmann
Keyword(s):  
Testicular Cancer ◽  
Hodgkin’S Lymphoma ◽  
Gene Knockout ◽  
Clinical Importance ◽  
Hodgkin's Lymphoma ◽  
Whole Genome ◽  
Cell Models ◽  
Core Component ◽  
Methyl Transferase ◽  
Low Levels

AbstractIt is of clinical importance to identify biomarkers predicting the efficacy of DNA damaging drugs (genotoxins) so that non-responders are not unduly exposed to the deleterious effects of otherwise inefficient drugs. Using a whole genome CRISPR/Cas9 gene knockout approach we have identified that low levels of ASH2L cause resistance to genotoxins. ASH2L is a core component of the H3K4 methyl transferase complex. We show that ASH2L absence decreases cell proliferation and favors DNA repair upon genotoxin exposure. The cell models we have used are derived from cancers currently treated either partially (Hodgkin’s lymphoma), or entirely (testicular cancer) with genotoxins. For such cancers, ASH2L levels could be used as a biomarker to predict the response to genotoxins. Our data also indicate that patients with low ASH2L expressing tumors do not develop resistance to ATR inhibitors. In these patients, such inhibitors may represent an alternative treatment to DNA damaging drugs.

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