Introduction Of Point Mutations Research Articles

Monoclonal Antibody Targeting the HA191/199 Region of H1N1 Influenza Virus Mediates the Damage of Neural Cells.

Vaccination is the most effective mean of preventing influenza virus infections. However, vaccination-induced adverse reactions of the nervous system, the causes of which are unknown, lead to concerns on the safety of influenza A vaccine. In this study, we used flow cytometry, cell ELISA, and immunofluorescence to find that H1-84 monoclonal antibody (mAb) against the191/199 region of the H1N1 influenza virus hemagglutinin (HA) protein binds to neural cells and mediates cell damage. Using molecular simulation software, such as PyMOL and PDB viewer, we demonstrated that the HA191/199 region maintains the overall structure of the HA head. Since the HA191/199 region cannot be removed from the HA structure, it has to be altered via introducing point mutations by site-directed mutagenesis. This will provide an innovative theoretical support for the subsequent modification the influenza A vaccine for increasing its safety.Electronic supplementary materialThe online version contains supplementary material available at 10.1134/S0006297921110109.

Genetic modifications of metallothionein enhance the tolerance and bioaccumulation of heavy metals in Escherichia coli

Metallothioneins (MTs) are low molecular weight cysteine-rich proteins that bind to metals. Owing to their high cysteine (Cys) content, MTs are effective mediators of heavy metal detoxification. To enhance the heavy metal binding ability of MT from the freshwater crab Sinopotamon henanense (ShMT), sequence-based multiple sequence alignment (MSA) and structure-based molecular docking simulation (MDS) were conducted in order to identify amino acid residues that could be mutated to bolster such metal-binding activity. Site-directed mutagenesis was then used to modify the primary structure of ShMT, and the recombinant proteins were further enhanced using the SUMO fusion expression system to yield SUMO-ShMT1, SUMO-ShMT2, and SUMO-ShMT3 harboring one-, two-, and three- point mutations, respectively. The resultant modified proteins were primarily expressed in a soluble form and exhibited the ability to readily bind to heavy metals. Importantly, these modified proteins exhibited significantly enhanced heavy metal binding capacities, and they improved Cd2+, Cu2+ and Zn2+ tolerance and bioaccumulation in Escherichia coli (E. coli) in a manner dependent upon the number of introduced point mutations (SUMO-ShMT3 > SUMO-ShMT2 > SUMO-ShMT1 > SUMO-ShMT > control). Indeed, E. coli cells harboring the pET28a-SUMO-ShMT3 expression vector exhibited maximal Cd2+, Cu2+, and Zn2+ bioaccumulation that was increased by 1.86 ± 0.02-, 1.71 ± 0.03-, and 2.13 ± 0.02-fold relative to that in E. coli harboring the pET28a-SUMO-ShMT vector. The present study offers a basis for the preparation of genetically engineered bacteria that are better able to bioaccumulate and tolerate heavy metals, thus providing a foundation for biological heavy metal water pollution treatment.

Enhancing Electrochemical Biosensor Selectivity with Engineered d-Amino Acid Oxidase Enzymes for d-Serine and d-Alanine Quantification.

d-Amino acid oxidase (DAAO) enzymes bind a range of d-amino acids with variable affinity. As such, the design of selective DAAO-based enzymatic biosensors remains a challenge for real-world biosensor application. Herein, a methodology for developing biosensors with varying substrate selectivity is presented. First, we address DAAO-based biosensor selectivity toward d-serine by introducing point mutations into DAAO using rational design. Next, the wild-type yeast DAAO (RgDAAO WT) and variants human DAAO W209R and yeast M213G are characterized for their selectivity and activity toward d-serine and d-alanine, the preferred DAAO substrates. The DAAO enzymes have been immobilized for final biosensor design, where they demonstrate selectivity comparable to free DAAO. The cross-linking procedure impacts on DAAO structure and function and the use of a regeneration strategy allows the biosensor response to be improved.

Enzymatic Activity of Glycosyltransferase Glt8d1 Promotes Human Glioblastoma Cells Migration

Glioblastoma (GBM) is the most aggressive primary brain tumor characterized by infiltrative migration of malignant glioma cells into the surrounding brain parenchyma. In this study, our analysis of GBM patient cohorts displayed significant higher expression of Glycosyltransferase 8 domain containing 1 ( GLT8D1 ) compared to normal brain tissue and could be associated with impaired patient survival. In vitro an increased GLT8D1 expression significantly enhanced migration of two different sphere-forming GBM cell lines. By in silico analysis we predicted the 3D-structure as well as the active site residues of GLT8D1. The introduction of point mutations in predicted active site reduced its glycosyltransferase activity in vitro and consequently impaired GBM tumor cell migration. Examination of GLT8D1 interaction partners by LC-MS/MS implied proteins associated with cytoskeleton and intracellular transport as potential substrates. In conclusion, we demonstrated that the enzymatic activity of glycosyltransferase GLT8D1 promotes GBM cell migration.

CRISPR Knock-in Designer: Automatic Oligonucleotide Design Software to Introduce Point Mutations by Gene Editing Methods

Abstract Background: Knock-in of precise point mutations into protein-coding genes is among the most important applications of Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9. Pre...

Development of Clostridium saccharoperbutylacetonicum as a Whole Cell Biocatalyst for Production of Chirally Pure (R)-1,3-Butanediol.

Chirally pure (R)-1,3-butanediol ((R)-1,3-BDO) is a valuable intermediate for the production of fragrances, pheromones, insecticides and antibiotics. Biotechnological production results in superior enantiomeric excess over chemical production and is therefore the preferred production route. In this study (R)-1,3-BDO was produced in the industrially important whole cell biocatalyst Clostridium saccharoperbutylacetonicum through expression of the enantio-specific phaB gene from Cupriavidus necator. The heterologous pathway was optimised in three ways: at the transcriptional level choosing strongly expressed promoters and comparing plasmid borne with chromosomal gene expression, at the translational level by optimising the codon usage of the gene to fit the inherent codon adaptation index of C. saccharoperbutylacetonicum, and at the enzyme level by introducing point mutations which led to increased enzymatic activity. The resulting whole cell catalyst produced up to 20 mM (1.8 g/l) (R)-1,3-BDO in non-optimised batch fermentation which is a promising starting position for economical production of this chiral chemical.

Strategies for Bacteriophage T5 Mutagenesis: Expanding the Toolbox for Phage Genome Engineering.

Phage genome editing is crucial to uncover the molecular mechanisms of virus infection and to engineer bacteriophages with enhanced antibacterial properties. Phage genetic engineering relies mostly on homologous recombination (HR) assisted by the targeted elimination of wild-type phages by CRISPR-Cas nucleases. These strategies are often less effective in virulent bacteriophages with large genomes. T5 is a virulent phage that infects Escherichia coli. We found that CRISPR-Cas9 system (type II-A) had ununiform efficacies against T5, which impairs a reliable use of CRISPR-Cas-assisted counterselection in the gene editing of T5. Here, we present alternative strategies for the construction of mutants in T5. Bacterial retroelements (retrons) proved to be efficient for T5 gene editing by introducing point mutations in the essential gene A1. We set up a protocol based on dilution-amplification-screening (DAS) of phage pools for mutant enrichment that was used to introduce a conditional mutation in another essential gene (A2), insert a new gene (lacZα), and construct a translational fusion of a late phage gene with a fluorescent protein coding gene (pb10-mCherry). The method should be applicable to other virulent phages that are naturally resistant to CRISPR/Cas nucleases.

Arpin Regulates Migration Persistence by Interacting with Both Tankyrases and the Arp2/3 Complex.

During cell migration, protrusion of the leading edge is driven by the polymerization of Arp2/3-dependent branched actin networks. Migration persistence is negatively regulated by the Arp2/3 inhibitory protein Arpin. To better understand Arpin regulation in the cell, we looked for its interacting partners and identified both Tankyrase 1 and 2 (TNKS) using a yeast two-hybrid screening and coimmunoprecipitation with full-length Arpin as bait. Arpin interacts with ankyrin repeats of TNKS through a C-terminal-binding site on its acidic tail, which overlaps with the Arp2/3-binding site. Arpin was found to dissolve the liquid–liquid phase separation of TNKS upon overexpression. To uncouple the interactions of Arpin with TNKS and Arp2/3, we introduced point mutations in the Arpin tail and attempted to rescue the increased migration persistence of the Arpin knockout cells using random plasmid integration or compensating knock-ins at the ARPIN locus. Arpin mutations impairing interactions with either Arp2/3 or TNKS were insufficient to fully abolish Arpin activity. Only the mutation that affected both interactions rendered Arpin completely inactive, suggesting the existence of two independent pathways, whereby Arpin controls the migration persistence.

Engineering and crystal structure of a monomeric FLT3 ligand variant.

The overarching paradigm for the activation of class III and V receptor tyrosine kinases (RTKs) prescribes cytokine-mediated dimerization of the receptor ectodomains and homotypic receptor-receptor interactions. However, structural studies have shown that the hematopoietic receptor FLT3, a class III RTK, does not appear to engage in such receptor-receptor contacts, despite its efficient dimerization by dimeric FLT3 ligand (FL). As part of efforts to better understand the intricacies of FLT3 activation, we sought to engineer a monomeric FL. It was found that a Leu27Asp substitution at the dimer interface of the cytokine led to a stable monomeric cytokine (FLL27D) without abrogation of receptor binding. The crystal structure of FLL27D at 1.65 Å resolution revealed that the introduced point mutation led to shielding of the hydrophobic footprint of the dimerization interface in wild-type FL without affecting the conformation of the FLT3 binding site. Thus, FLL27D can serve as a monomeric FL variant to further interrogate the assembly mechanism of extracellular complexes of FLT3 in physiology and disease.

A procedure to introduce point mutations into the Rubisco large subunit gene in wild-type plants.

Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Rubisco is a key target for improvement as it plays a central role in carbon fixation during photosynthesis and is remarkably inefficient. Introduction of mutations to the chloroplast-encoded Rubisco large subunit rbcL is of particular interest for improving the catalytic activity and efficiency of the enzyme. However, manipulation of rbcL is hampered by its location in the plastome, with many species recalcitrant to plastome transformation, and by the plastid's efficient repair system, which can prevent effective maintenance of mutations introduced with homologous recombination. Here we present a system where the introduction of a number of silent mutations into rbcL within the model plant Nicotiana tabacum facilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild-type N. tabacum with stable point mutations within rbcL in 40% of the transformants, allowing assessment of the effect of these mutations on Rubisco assembly and activity. With further optimization the approach offers a viable way forward for mutagenic testing of Rubisco function in planta within tobacco and modification of rbcL in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast-encoded genes.

A Salmonella Typhi RNA thermosensor regulates virulence factors and innate immune evasion in response to host temperature.

Sensing and responding to environmental signals is critical for bacterial pathogens to successfully infect and persist within hosts. Many bacterial pathogens sense temperature as an indication they have entered a new host and must alter their virulence factor expression to evade immune detection. Using secondary structure prediction, we identified an RNA thermosensor (RNAT) in the 5' untranslated region (UTR) of tviA encoded by the typhoid fever-causing bacterium Salmonella enterica serovar Typhi (S. Typhi). Importantly, tviA is a transcriptional regulator of the critical virulence factors Vi capsule, flagellin, and type III secretion system-1 expression. By introducing point mutations to alter the mRNA secondary structure, we demonstrate that the 5' UTR of tviA contains a functional RNAT using in vitro expression, structure probing, and ribosome binding methods. Mutational inhibition of the RNAT in S. Typhi causes aberrant virulence factor expression, leading to enhanced innate immune responses during infection. In conclusion, we show that S. Typhi regulates virulence factor expression through an RNAT in the 5' UTR of tviA. Our findings demonstrate that limiting inflammation through RNAT-dependent regulation in response to host body temperature is important for S. Typhi's "stealthy" pathogenesis.

MAVERICC: Marker-free Vaccinia Virus Engineering of Recombinants through in vitro CRISPR/Cas9 Cleavage

Vaccinia virus (VACV)-based vectors are in extensive use as vaccines and cancer immunotherapies. VACV engineering has traditionally relied on homologous recombination between a parental viral genome and a transgene-bearing transfer plasmid, an inefficient process that necessitates the use of a selection or screening marker to isolate recombinants. Recent extensions of this approach have sought to enhance the recovery of transgene-bearing viruses through the use of CRISPR-Cas9 engineering to cleave the viral genome in infected cells. However, these methods do not completely eliminate the generation of WT viral progeny and thus continue to require multiple rounds of viral propagation and plaque purification. Here, we describe MAVERICC (marker-free vaccinia virus engineering of recombinants through in vitroCRISPR/Cas9 cleavage), a new strategy to engineer recombinant VACVs in a manner that overcomes current limitations. MAVERICC also leverages the CRISPR/Cas9 system but requires no markers and yields essentially pure preparations of the desired recombinants in a single step. We used this approach to introduce point mutations, insertions, and deletions at multiple locations in the VACV genome, both singly and in combination. The efficiency and versatility of MAVERICC make it an ideal choice for generating mutants and mutant libraries at arbitrarily selected locations in the viral genome to build complex VACV vectors, effect vector improvements, and facilitate the study of poxvirus biology.

Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis.

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

Base and Prime Editing Technologies for Blood Disorders.

Nuclease-based genome editing strategies hold great promise for the treatment of blood disorders. However, a major drawback of these approaches is the generation of potentially harmful double strand breaks (DSBs). Base editing is a CRISPR-Cas9-based genome editing technology that allows the introduction of point mutations in the DNA without generating DSBs. Two major classes of base editors have been developed: cytidine base editors or CBEs allowing C>T conversions and adenine base editors or ABEs allowing A>G conversions. The scope of base editing tools has been extensively broadened, allowing higher efficiency, specificity, accessibility to previously inaccessible genetic loci and multiplexing, while maintaining a low rate of Insertions and Deletions (InDels). Base editing is a promising therapeutic strategy for genetic diseases caused by point mutations, such as many blood disorders and might be more effective than approaches based on homology-directed repair, which is moderately efficient in hematopoietic stem cells, the target cell population of many gene therapy approaches. In this review, we describe the development and evolution of the base editing system and its potential to correct blood disorders. We also discuss challenges of base editing approaches–including the delivery of base editors and the off-target events–and the advantages and disadvantages of base editing compared to classical genome editing strategies. Finally, we summarize the recent technologies that have further expanded the potential to correct genetic mutations, such as the novel base editing system allowing base transversions and the more versatile prime editing strategy.

Knockdown of the plastid-encoded acetyl-CoA carboxylase gene uncovers functions in metabolism and development.

De novo fatty acid biosynthesis in plants relies on a prokaryotic-type acetyl-CoA carboxylase (ACCase) that resides in the plastid compartment. The enzyme is composed of four subunits, one of which is encoded in the plastid genome, whereas the other three subunits are encoded by nuclear genes. The plastid gene (accD) encodes the β-carboxyltransferase subunit of ACCase and is essential for cell viability. To facilitate the functional analysis of accD, we pursued a transplastomic knockdown strategy in tobacco (Nicotiana tabacum). By introducing point mutations into the translational start codon of accD, we obtained stable transplastomic lines with altered ACCase activity. Replacement of the standard initiator codon AUG with UUG strongly reduced AccD expression, whereas replacement with GUG had no detectable effects. AccD knockdown mutants displayed reduced ACCase activity, which resulted in changes in the levels of many but not all species of cellular lipids. Limiting fatty acid availability caused a wide range of macroscopic, microscopic, and biochemical phenotypes, including impaired chloroplast division, reduced seed set, and altered storage metabolism. Finally, while the mutants displayed reduced growth under photoautotrophic conditions, they showed exaggerated growth under heterotrophic conditions, thus uncovering an unexpected antagonistic role of AccD activity in autotrophic and heterotrophic growth.

Targeted mutagenesis in human iPSCs using CRISPR genome-editing tools.

Mutagenesis studies have rapidly evolved in the era of CRISPR genome editing. Precise manipulation of genes in human induced pluripotent stem cells (iPSCs) allows biomedical researchers to study the physiological functions of individual genes during development. Furthermore, such genetic manipulation applied to patient-specific iPSCs allows disease modeling, drug screening and development of therapeutics. Although various genome-editing methods have been developed to introduce or remove mutations in human iPSCs, comprehensive strategic designs taking account of the potential side effects of CRISPR editing are needed. Here we present several novel and highly efficient strategies to introduce point mutations, insertions and deletions in human iPSCs, including step-by-step experimental protocols. These approaches involve the application of drug selection for effortless clone screening and the generation of a wild type control strain along with the mutant. We also present several examples of application of these strategies in human iPSCs and show that they are highly efficient and could be applied to other cell types.

Conformational switch that induces GDP release from Gi

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are composed of α, β, and γ subunits. Gα switches between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active states, and Gβγ interacts with the GDP-bound state. The GDP-binding regions are composed of two sites: the phosphate-binding and guanine-binding regions. The turnover of GDP and GTP is induced by guanine nucleotide-exchange factors (GEFs), including G protein-coupled receptors (GPCRs), Ric8A, and GIV/Girdin. However, the key structural factors for stabilizing the GDP-bound state of G proteins and the direct structural event for GDP release remain unclear. In this study, we investigated structural factors affecting GDP release by introducing point mutations in selected, conserved residues in Gαi3. We examined the effects of these mutations on the GDP/GTP turnover rate and the overall conformation of Gαi3 as well as the binding free energy between Gαi3 and GDP. We found that dynamic changes in the phosphate-binding regions are an immediate factor for the release of GDP.

Rice Gene Targeting by Homologous Recombination with Positive-Negative Selection Strategy.

Gene targeting (GT) is a technique that alter the structure of the specific genes at their original loci in the genome by homologous recombination (HR). It plays an important role in functional genomics because it enables precise modification of the endogenous genes into desired forms such as knockout, knock-in, introduction of point mutations, as well as generation of fusion genes. Also, site-directed mutagenesis by GT can also be applied as an excellent technique for molecular breeding and gene therapy, because it can directly reflect the knowledge acquired from functional genomics. In this section, we introduce well-established GT procedure in rice in combination with positive-negative-selection (PNS) strategy.

Rapid Vector Construction and Assessment of BE3 and Target-AID C to T Base Editing Systems in Rice Protoplasts.

CRISPR-Cas9 has revolutionized the field of genome engineering. Base editing, a new genome editing strategy, was recently developed to engineer nucleotide substitutions. DNA base editing systems use a catalytically impared Cas nuclease together with a nucleobase deaminase enzyme to specifically introduce point mutations without generating double-stranded breaks, which provide huge potential in crop improvement. Here, we describe fast and efficient preparation of user-friendly C to T base editors, BE3, and Target-AID. Presented are detailed protocols for T-DNA vector preparation with BE3 or modified Target-AID base editor based on Gateway assembly and efficiency assessment of base editing through a rice protoplast transient expression system.

The availability of neither D2 nor CP43 limits the biogenesis of photosystem II in tobacco.

The pathway of photosystem II (PSII) assembly is well understood, and multiple auxiliary proteins supporting it have been identified, but little is known about rate-limiting steps controlling PSII biogenesis. In the cyanobacterium Synechocystis PCC6803 and the green alga Chlamydomonas reinhardtii, indications exist that the biosynthesis of the chloroplast-encoded D2 reaction center subunit (PsbD) limits PSII accumulation. To determine the importance of D2 synthesis for PSII accumulation in vascular plants and elucidate the contributions of transcriptional and translational regulation, we modified the 5′-untranslated region of psbD via chloroplast transformation in tobacco (Nicotiana tabacum). A drastic reduction in psbD mRNA abundance resulted in a strong decrease in PSII content, impaired photosynthetic electron transport, and retarded growth under autotrophic conditions. Overexpression of the psbD mRNA also increased transcript abundance of psbC (the CP43 inner antenna protein), which is co-transcribed with psbD. Because translation efficiency remained unaltered, translation output of pbsD and psbC increased with mRNA abundance. However, this did not result in increased PSII accumulation. The introduction of point mutations into the Shine–Dalgarno-like sequence or start codon of psbD decreased translation efficiency without causing pronounced effects on PSII accumulation and function. These data show that neither transcription nor translation of psbD and psbC are rate-limiting for PSII biogenesis in vascular plants and that PSII assembly and accumulation in tobacco are controlled by different mechanisms than in cyanobacteria or in C. reinhardtii.