The impact of Cas9-mediated mutagenesis of genes encoding potato starch-branching enzymes on starch structural properties and in vitro digestibility.

The digestibility of starch is affected by amylose content, and increasing amylopectin chain length which can be manipulated by alterations to genes encoding starch-branching enzymes (SBEs). We investigated the impact of Cas9-mediated mutagenesis of SBEs in potato on starch structural properties and digestibility. Four potato starches with edited SBE genes were tested. One lacked SBE1 and SBE2, two lacked SBE2 and had reduced SBE1, and one had reduced SBE2 only. Starch structure and thermal properties were characterised by DSC and XRD. The impact of different thermal treatments on digestibility was studied using an in vitro digestion protocol. All native potato starches were resistant to digestion, and all gelatinised starches were highly digestible. SBE modified starches had higher gelatinisation temperatures than wild type potatoes and retrograded more rapidly. Gelatinisation and 18 h of retrogradation, increased gelatinisation enthalpy, but this did not translate to differences in digestion. Following 7 days of retrogradation, starch from three modified SBE starch lines was less digestible than starch from wild-type potatoes, likely due to the recrystallisation of the long amylopectin chains. Our results indicate that reductions in SBE in potato may be beneficial to health by increasing the amount of fibre reaching the colon after retrogradation.

Analysis of oil accumulation mechanisms in plasma induced mutant Scenedesmus strains compared to original Scenedesmus strains.

Scenedesmus sp. is a species of the Scenedesmus genus within the phylum Chlorophyta, commonly found as a planktonic algal species in freshwater and known for its rapid growth rate. This study employs room-temperature, atmospheric-pressure plasma mutagenesis for the breeding of Scenedesmus sp., utilizing transcriptomic analysis to investigate the biosynthesis mechanism of triglycerides. Further analysis of differentially expressed genes in transcriptome by measuring the macroscopic biological indicators of mutant and original algal strains. The findings of the study suggest that the mutant strain's photosynthesis has been enhanced, leading to improved light energy utilization and CO2 fixation, thereby providing more carbon storage and energy for biomass and lipid production. The intensification of glycolysis and the TCA (tricarboxylic acid) cycle results in a greater shift in carbon flux towards lipid accumulation. An elevated expression level of related enzymes in starch and protein degradation pathways may enhance acetyl CoA accumulation, facilitating a larger substrate supply for fatty acid production and thereby increasing lipid yield.

A user-friendly CRISPR/Cas9 system for mutagenesis of Neurospora crassa.

As a widely used eukaryotic model organism, Neurospora crassa offers advantages in genetic studies due to its diverse biology and rapid growth. Traditional genetic manipulation methods, such as homologous recombination, require a considerable amount of time and effort. In this study, we present an easy-to-use CRIPSR/Cas9 system for N. crassa, in which the cas9 sequence is incorporated into the fungal genome and naked guide RNA is introduced via electroporation. Our approach eliminates the need for constructing multiple vectors, speeding up the mutagenesis process. Using cyclosporin-resistant-1 (csr-1) as a selectable marker gene, we achieved 100% editing efficiency under selection conditions. Furthermore, we successfully edited the non-selectable gene N-acylethanolamine amidohydrolase-2 (naa-2), demonstrating the versatility of the system. Combining gRNAs targeting csr-1 and naa-2 simultaneously increased the probability of finding mutants carrying the non-selectable mutation. The system is not only user-friendly but also effective, providing a rapid and efficient method for generating loss-of-function mutants in N. crassa compared to traditional methods.

[Construction of a Kluyveromyces lactis strain with multi-copy integration for enhanced bovine chymosin production by CRISPR/Cas9 and UV mutagenesis].

Bovine chymosin is an essential food enzyme widely used in cheese production in the dairy industry. This study used a codon-optimized prochymosin gene to construct an expression cassette for extracellular expression of bovine chymosin in Kluyveromyces lactis. After integration of the prochymosin gene into the host cell genome, the single-copy integration strain KLUcym showed the clotting activity of 40 U/mL in a shake flask. The CRISPR/Cas9 system was employed to delete amdS and construct the double-copy integration strain and triple-copy integration strain, which achieved the clotting activities of 70 U/mL and 78 U/mL in shake flasks, separately. Subsequently, multiple rounds of UV mutagenesis were performed on the double-copy strain KLUcymD, and a recombinant K. lactis strain with a high yield of bovine chymosin was obtained. This strain achieved the clotting activity of 270 U/mL in a shake flask and 600 U/mL in a 5 L bioreactor after 76 h. In summary, we construct a strain KLUcymD-M2 for high production of bovine chymosin, which lays a foundation of industrial fermentation.

[Breeding of L-tryptophan high-yield strain by ARTP mutagenesis and high-throughput screening].

L-tryptophan is an indispensable essential amino acid with a wide range of applications, which leads to a high demand. Accordingly, the production of L-tryptophan becomes a much-anticipated direction in research and industrial development. While irrational mutagenesis is an effective means to breed industrial strains, how to screen the strains with desirable phenotypes is still a major challenge. In order to improve the efficiency and accuracy of screening L-tryptophan high-yield strains, we used atmospheric and room temperature plasma mutagenesis to construct a random mutant library and then combined it with high-throughput screening in deep-well plates. Using a pseudo-fluorescent protein sensor capable of responding specifically to L-tryptophan, we successfully screened out a strain producing L-tryptophan at a high yield from a random mutagenesis library. The fermentation with the strain in shake flasks produced L-tryptophan at a yield of 1.99 g/L, which was 41.77% higher than that of the starting strain. Finally, the mechanism of high yield of the strain was deciphered by comparative genomics and transcriptomics. The above strategies provide a solid research foundation for further selection and development of high quality L-tryptophan producing strains.

Computational Mutagenesis and Inhibition of Staphylococcus aureus AgrA LytTR Domain Using Phenazine Scaffolds: Insight From a Biophysical Study.

Biofilm formation by Staphylococcus aureus is a major challenge in clinical settings due to its role in persistent infections. The AgrA protein, a key regulator in biofilm development, is a promising target for therapeutic intervention. This study investigates the antibiofilm potential of halogenated phenazine compounds by targeting AgrA and explores their molecular interactions to provide insights for drug development. We employed molecular docking, molecular dynamics simulations, and computational mutagenesis to evaluate the binding of halogenated phenazine compounds (C1 to C7, HP, and HP-14) to AgrA. Binding free energy analysis was performed to assess the affinity of these compounds for the AgrA-DNA complex. Additionally, the impact of these compounds on AgrA's structural conformation and salt bridge interactions was examined. The binding-free energy analysis revealed that all compounds enhance binding affinity compared to the Apo form of AgrA, which has a ΔGbind of -80.75 kcal/mol. The strongest binding affinities were observed with compounds C7 (-113.84 kcal/mol), HP-14 (-115.23 kcal/mol), and HP (-112.28 kcal/mol), highlighting their effectiveness. Molecular dynamics simulations demonstrated that these compounds bind at the hydrophobic cleft of AgrA, disrupting essential salt bridge interactions between His174-Glu163 and His174-Glu226. This disruption led to structural conformational changes and reduced DNA binding affinity, aligning with experimental findings on biofilm inhibition. The halogenated phenazine compounds effectively inhibit biofilm formation by targeting AgrA, disrupting its DNA-binding function. The study supports the potential of these compounds as antibiofilm agents and provides a foundation for rational drug design targeting the AgrA-DNA interaction. Future research should focus on further optimizing these lead compounds and exploring additional active sites on AgrA to develop novel treatments for biofilm-associated infections.

Mutating a flexible region of the RSV F protein can stabilize the prefusion conformation.

The respiratory syncytial virus (RSV) fusion (F) glycoprotein is highly immunogenic in its prefusion (pre-F) conformation. However, the protein is unstable, and its conformation must be stabilized for it to function effectively as an immunogen in vaccines. We present a mutagenesis strategy to arrest the RSV F protein in its pre-F state by blocking localized changes in protein structure that accompany large-scale conformational rearrangements. We generated a series of mutants and screened them in vitro to assess their potential for forming a stable pre-F. In animals, the immunogenicity of a representative mutant F protein, with a conformation confirmed by cryo-electron microscopy, elicited levels of neutralizing antibodies and protection against RSV-induced lung damage that were comparable to those of DS-Cav1, a pre-F used in a licensed vaccine.

A machine learning enhanced EMS mutagenesis probability map for efficient identification of causal mutations in Caenorhabditis elegans.

Chemical mutagenesis-driven forward genetic screens are pivotal in unveiling gene functions, yet identifying causal mutations behind phenotypes remains laborious, hindering their high-throughput application. Here, we reveal a non-uniform mutation rate caused by Ethyl Methane Sulfonate (EMS) mutagenesis in the C. elegans genome, indicating that mutation frequency is influenced by proximate sequence context and chromatin status. Leveraging these factors, we developed a machine learning enhanced pipeline to create a comprehensive EMS mutagenesis probability map for the C. elegans genome. This map operates on the principle that causative mutations are enriched in genetic screens targeting specific phenotypes among random mutations. Applying this map to Whole Genome Sequencing (WGS) data of genetic suppressors that rescue a C. elegans ciliary kinesin mutant, we successfully pinpointed causal mutations without generating recombinant inbred lines. This method can be adapted in other species, offering a scalable approach for identifying causal genes and revitalizing the effectiveness of forward genetic screens.

DNA mismatch repair controls the mutagenicity of Polymerase ζ-dependent translesion synthesis at methylated guanines.

By replicating damaged nucleotides, error-prone DNA translesion synthesis (TLS) enables the completion of replication, albeit at the expense of fidelity. TLS of helix-distorting DNA lesions, that usually have reduced capacity of basepairing, comprises insertion opposite the lesion followed by extension, the latter in particular by polymerase ζ (Pol ζ). However, little is known about involvement of Pol ζ in TLS of non- or poorly-distorting, but miscoding, lesions such as O6-methyldeoxyguanosine (O6-medG). Using purified Pol ζ we describe that the enzyme can misincorporate thymidine opposite O6-medG and efficiently extend from terminal mismatches, suggesting its involvement in the mutagenicity of O6-medG. Surprisingly, O6-medG lesions induced by the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) appeared more, rather than less, mutagenic in Pol ζ-deficient mouse embryonic fibroblasts (MEFs) than in wild type MEFs. This suggested that in vivo Pol ζ participates in non-mutagenic TLS of O6-medG. However, we found that the Pol ζ-dependent misinsertions at O6-medG lesions are efficiently corrected by DNA mismatch repair (MMR), which masks the error-proneness of Pol ζ. We also found that the MNNG-induced mutational signature is determined by the adduct spectrum, and modulated by MMR. The signature mimicked single base substitution signature 11 in the catalogue of somatic mutations in cancer, associated with treatment with the methylating drug temozolomide. Our results unravel the individual roles of the major contributors to methylating drug-induced mutagenesis. Moreover, these results warrant caution as to the classification of TLS as mutagenic or error-free based on in vitro data or on the analysis of mutations induced in MMR-proficient cells.

Visualization, Fate Mapping, Ablation, and Mutagenesis of Microglia in the Mouse Brain.

Since the classical studies of Pío del Río-Hortega, microglia research has come a long way. In particular, recent advances in bulk and single-cell (sc) transcriptomics have yielded many fascinating new insights into these intriguing immune cells at the interface with the central nervous system (CNS), both in small animal models and human samples. In parallel, tools developed by advanced mouse genetics have revealed the unique ontogeny of microglia and their striking dynamic interactions with other cells in the brain parenchyma. In this chapter, we will discuss various applications of the Cre/loxP-based approach that have enabled the study of microglia in their physiological context of the mouse brain. We will highlight selected key findings that have shaped our current understanding of these cells and discuss the technical intricacies of the Cre/loxP approach and some remaining challenges.

ARTP mutagenesis of Aureobasidium pullulans RM1603 for high pullulan production and transcriptome analysis of mutants.

Pullulan is a microbial exopolysaccharide produced by Aureobasidium spp. with excellent physical and chemical properties, resulting in great application value. In this study, a novel strain RM1603 of Aureobasidium pullulans with high pullulan production of 51.0 ± 1.0 g·L- 1 isolated from rhizosphere soil was subjected to atmospheric and room temperature plasma (ARTP) mutagenesis, followed by selection of mutants to obtain pullulan high-producing strains. Finally, two mutants Mu0816 and Mu1519 were obtained, with polysaccharide productions of 58.7 ± 0.8 and 60.0 ± 0.8 g∙L- 1 after 72-h fermentation, representing 15.1 and 17.6% increases compared with the original strain, respectively. Transcriptome analysis of the two mutants and the original strain revealed that the high expression of α/ß-hydrolase (ABHD), α-amylase (AMY1), and sugar porter family MFS transporters (SPF-MFS) in the mutants may be related to the synthesis and secretion of pullulan. These results demonstrated the effectiveness of ARTP mutagenesis in A. pullulans, providing a basis for the investigation of genes related to pullulan synthesis and secretion.

MADDD-seq, a novel massively parallel sequencing tool for simultaneous detection of DNA damage and mutations.

Our genome is exposed to a wide variety of DNA-damaging agents. If left unrepaired, this damage can be converted into mutations that promote carcinogenesis or the development of genetically inherited diseases. As a result, researchers and clinicians require tools that can detect DNA damage and mutations with exceptional sensitivity. In this study, we describe a massively parallel sequencing tool termed Mutation And DNA Damage Detection-seq (MADDD-seq) that is capable of detecting O6-methyl guanine lesions and mutations simultaneously, with a single assay. To illustrate the dual capabilities of MADDD-seq, we treated WT and DNA repair deficient yeast cells with the DNA-damaging agent MNNG and tracked DNA lesions and mutations over a 24-h time period. This approach allowed us to identify thousands of DNA adducts and mutations in a single sequencing run and gain deep insight into the kinetics of DNA repair and mutagenesis.

FAM72A degrades UNG2 through the GID/CTLH complex to promote mutagenic repair during antibody maturation.

A diverse antibody repertoire is essential for humoral immunity. Antibody diversification requires the introduction of deoxyuridine (dU) mutations within immunoglobulin genes to initiate somatic hypermutation (SHM) and class switch recombination (CSR). dUs are normally recognized and excised by the base excision repair (BER) protein uracil-DNA glycosylase 2 (UNG2). However, FAM72A downregulates UNG2 permitting dUs to persist and trigger SHM and CSR. How FAM72A promotes UNG2 degradation is unknown. Here, we show that FAM72A recruits a C-terminal to LisH (CTLH) E3 ligase complex to target UNG2 for proteasomal degradation. Deficiency in CTLH complex components result in elevated UNG2 and reduced SHM and CSR. Cryo-EM structural analysis reveals FAM72A directly binds to MKLN1 within the CTLH complex to recruit and ubiquitinate UNG2. Our study further suggests that FAM72A hijacks the CTLH complex to promote mutagenesis in cancer. These findings show that FAM72A is an E3 ligase substrate adaptor critical for humoral immunity and cancer development.

Biochemical foundation of the aroma and antioxidant activity of Indian traditional rice landrace Maharaji and the effect of radiation-induced mutagenesis on its metabolome.

Maharaji rice, an aromatic variety with medium slender grains, is traditionally cultivated in the central regions of India. This study aimed to identify the biochemical compounds responsible for Maharaji rice's distinctive fragrance and enhance its agro-morphological traits through mutation breeding. Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) analysis, forty major metabolites were identified which may be responsible for its characteristic aroma. The bioactive compounds included terpenes, flavonoids, and amino acids. Maharaji brown rice extract exhibited potent radical scavenging activity. Radiation-induced mutation breeding improved the agro-morphological traits and also triggered biochemical diversification in different mutants. Maharaji Mutant-2 exhibited improved aroma due to higher abundance of aromatic compounds, improved yield and morphological characters as compared to the parent. This study, for the first time identifies the compounds associated with the characteristic aroma of Maharaji rice. Global metabolomics may, therefore, expedite the selection of mutants with suitable aroma and desirable biological properties.

Machine learning-guided multi-site combinatorial mutagenesis enhances the thermostability of pectin lyase.

Enhancing the thermostability of enzymes is crucial for industrial applications. Methods such as directed evolution are often limited by the huge sequence space and combinatorial explosion, making it difficult to obtain optimal mutants. In recent years, machine learning (ML)-guided protein engineering has become an attractive tool because of its ability to comprehensively explore the sequence space of enzymes and discover superior mutants. This study employed ML to perform combinatorial mutation design on the pectin lyase PMGL-Ba from Bacillus licheniformis, aiming to improve its thermostability. First, 18 single-point mutants with enhanced thermostability were identified through semi-rational design. Subsequently, the initial library containing a small number of low-order mutants was utilized to construct an ML model to explore the combinatorial sequence space (theoretically 196,608 mutants) of single-point mutants. The results showed that the ML-predicted second library was successfully enriched with highly thermostable combinatorial mutants. After one iteration of learning, the best-performing combinatorial mutant in the third library, P36, showed a 67-fold and 39-fold increase in half-life at 75 °C and 80 °C, respectively, as well as a 2.1-fold increase in activity. Structural analysis and molecular dynamics simulations provided insights into the improved performance of the engineered enzyme.

Comparison of Mutations Induced by Different Doses of Fast-Neutron Irradiation in the M1 Generation of Sorghum (Sorghum bicolor).

Sorghum is an important C4 crop with various food and nonfood uses. Although improvements through hybridization and selection have been exploited, the introduction of genetic variation and the development of new genotypes in sorghum are still limited. Fast-neutron (FN) mutagenesis is a very effective method for gene functional studies and to create genetic variability. However, the full spectrum of FN-induced mutations in sorghum is poorly understood. To address this, we generated an FN-induced mutant population from the inbred line 'BTx623' and sequenced 40 M1 seedlings to evaluate the mutagenic effects of FNs on sorghum. The results show that each line had an average of 43.7 single-base substitutions (SBSs), 3.7 InDels and 35.15 structural variations (SVs). SBSs accounted for approximately 90.0% of the total number of small mutations. Among the eight treatment groups, FN irradiation at a dose of 19 Gy generated the highest number of mutations. The ratio of transition/transversion ranged from 1.77 to 2.21, and the G/C to A/T transition was the most common substitution in all mutant lines. The distributions of the identified SBSs and InDels were similar and uneven across the genome. An average of 3.63 genes were mutated in each mutant line, indicating that FN irradiation resulted in a suitable density of mutated genes, which can be advantageous for improving elite material for one specific or a few traits. These results provide a basis for the selection of the suitable dose of mutagen and new genetic resources for sorghum breeding.

Characterization and subcellular localization of selenium in Limosilactobacillus fermentum Ln-9 obtained by intense pulsed light-ultraviolet combined mutagenesis.

Lactic acid bacteria (LAB) can convert inorganic selenium (Se) to organic Se and elemental forms with low toxicity and high bioavailability, but a comprehensive Se analysis of Se-enriched LAB is lacking. In this study, Limosilactobacillus fermentum Ln-9 was obtained by intense pulsed light-ultraviolet combined mutagenesis, and its characteristics and subcellular localization of Se were analyzed. The results displayed that Ln-9 accumulated 3.03 times Se that of the original strain. Under optimal fermentation conditions, the total Se content of Se-enriched Ln-9 (SeLn-9) reached 12.16 mg/g with 96.34% contained in Se nanoparticles (SeNPs), which was much higher than that of organic macromolecules. Furthermore, SeNPs were mainly localized outside the cell, Se-proteins were in the membrane and cytoplasmic fractions, and Se-polysaccharides were in the membrane fraction. Besides, SeLn-9 maintained a good morphology and gastrointestinal tolerance and had an enhanced antioxidant capacity. These findings make Ln-9 promising for applications in the food industry.

Obtaining more contaminant-resistant variants from a native Chlorella vulgaris strain.

Cildáñez stream (in Matanza-Riachuelo basin, Buenos Aires) is one of the most polluted watercourses of Argentina, containing a mixed contamination from agricultural and industrial wastes. The application of water bioremediation processes for this kind of effluent will require microorganisms with a high tolerance to contamination. In this sense, obtaining higher contaminant-resistant microalgae lines is widely desired. In this study, adaptive laboratory evolution (ALE) and random mutagenesis were used to obtain Chlorella vulgaris LMPA-40 strains adapted to grow in polluted water from the Cildáñez stream. The ALE process was performed by 22 successive subcultures under selective pressure (Cildáñez wastewater alone or with the addition of phenol or H2O2) while random mutagenesis was performed with UV-C radiation at 275nm. Not all the cell lines obtained after ALE could adapt enough to overcome the stress caused by the Cildáñez wastewater, indicating that the process is quite random and depends on the stressor used. The best results were obtained for the Cildáñez wastewater adapted cells (Cild 3 strain) that were more resistant than the original strain. The concentration of protein, Chlorophyll A, Chlorophyll B, and carotenoids in the Cild 3 ALE evolved strain was higher than that of the control strain. However, this strain exhibited half of the lipid content compared to the same control strain. Interestingly, these alterations and the acquired tolerance may be reversed over time during storage. These findings suggest that the acquisition of novel cell lines could not be permanent, a fact that must be considered for future trials.

Engineering Oncogenic Hotspot Mutations on SF3B1 via CRISPR-Directed PRECIS Mutagenesis.

SF3B1 is the most recurrently mutated RNA splicing gene in cancer. However, research of its pathogenic role has been hindered by a lack of disease-relevant cell line models. Here, our study compared four genome engineering platforms to establish SF3B1 mutant cell lines: CRISPR-Cas9 editing, AAV homology-directed repair editing, base editing (ABEmax, ABE8e), and prime editing (PE2, PE3, PE5max). We showed that prime editing via PE5max achieved the most efficient SF3B1 K700E editing across a wide range of cell lines. Our approach was further refined by coupling prime editing with a fluorescent reporter that leverages a SF3B1 mutation-responsive synthetic intron to mark successfully edited cells. By applying this approach, called prime editing coupled intron-assisted selection (PRECIS), we introduced the K700E hotspot mutation into two chronic lymphocytic leukemia cell lines, HG-3 and MEC-1. We demonstrated that our PRECIS-engineered cells faithfully recapitulate known mutant SF3B1 phenotypes, including altered splicing, copy number variations, and cell-growth defect. Moreover, we discovered that the SF3B1 mutation can cause the loss of Y chromosome in chronic lymphocytic leukemia. Our results showcase that PRECIS is an efficient and generalizable method for engineering genetically faithful SF3B1 mutant models. Our approach provides new insights on the role of SF3B1 mutation in cancer and enables the generation of SF3B1 mutant cell lines in relevant cellular context. SIGNIFICANCE: This study developed an approach that can reliably and efficiently engineer SF3B1 mutation into different cellular contexts, thereby revealing novel roles of SF3B1 mutation in driving aberrant splicing, clonal evolution, and genome instability.

MutaT7GDE: A Single Chimera for the Targeted, Balanced, Efficient, and Processive Installation of All Possible Transition Mutations In Vivo.

Deaminase-T7 RNA polymerase fusion (MutaT7) proteins are a growing class of synthetic biology tools used to diversify target genes during in vivo laboratory evolution. To date, MutaT7 chimeras comprise either a deoxyadenosine or deoxycytidine deaminase fused to a T7 RNA polymerase. Their expression drives targeted deoxyadenosine-to-deoxyguanosine or deoxycytidine-to-deoxythymidine mutagenesis, respectively. Here, we repurpose recently engineered substrate-promiscuous general deaminases (GDEs) to establish a substantially simplified system based on a single chimeric enzyme capable of targeting both deoxyadenosine and deoxycytidine. We assess on- and off-target mutagenesis, strand and context preference, and parity of deamination for four different MutaT7GDE constructs. We identify a single chimera that installs all possible transition mutations more efficiently than preexisting, more cumbersome MutaT7 tools. The optimized MutaT7GDE chimera reported herein is a next-generation hypermutator capable of mediating efficient and uniform target-gene diversification during in vivo directed evolution.