Mutagenesis Techniques Research Articles

DIVERSITY OF THE PUMPKIN COLLECTION AND ITS HEREDITARY POTENTIAL. RESULTS AND PROSPECTS OF BREEDING PRACTICE

Background. Pumpkin is an important food, feed and medicinal crop. It is widely used by national cuisines in many countries and utilized as raw material for canning industry and in pharmacy to prepare drugs. The Kuban Experiment Station of VIR, where the collection of pumpkin genetic resources is maintained and regenerated for long-term storage in the genebank, conducts comprehensive research work to analyze the genetic diversity of pumpkin accessions and disclose the potential of its hereditary variation.Objective. The aim of this study was to screen various species from the global pumpkin collection and identify sources of traits valuable for breeders in order to develop promising cultivars and hybrids.Materials and methods. The pumpkin collection was studied and the breeding work was performed using the guidelines worked out by VIR’s scientists. The work included botanical description, study of variability and inheritance of traits, hybridization, inbreeding, mutagenesis, and different selection techniques (individual, group, individual/ family, and mass selection). In the first stages of the study, inbreeding was employed to select best plants from the collection in order to set up a trait-specific collection and incorporate its accessions into the hybridization process. Self-pollination was usedfor several years to obtain self-pollinated lines, sufficiently uniform in the selected trait. The hybrid progenies from crosses between parental forms were analyzed to throw light on the inheritance of major traits and their genetic control. The accessions were assessed for their earliness, productivity and quality. Evaluation of disease resistance was carried out in the field under natural infection pressure.Results and discussion. Targeted breeding efforts resulted in releasing cultivars of Cucurbita pepo L., C. maxima Duch. and C. moschata Duch. ex. Poir. having different maturation schedules and approved for cultivation in various regions of Russia: ‘Kustovaya oranzhevaya’, ‘Lechebnaya’, ‘Kustovaya zolotaya’, ‘Malyshka’, ‘Matreshka’, ‘Krasavitsa’, Zimnyaya sladkaya’, ‘Zhemchuzhina’, ‘Yantarnaya’ and ‘Mariya’. These cultivars are in demand among both agricultural producers and individual growers. New sources of valuable morphobiological and agronomic characters have been identified as useful for modern plant breeding trends: KL 625, ZhZM 692, L-180, L-193, KPL 168, KL 568 and KL 570. New bushy lines of C. maxima and C. moschata selected from cultivar and hybrid populations are now tested in an elite test nursery in order to submit the best of them to the State Variety Trials.

Functional analysis of six uncharacterised mutations in LDLR gene

Background and aimsFamilial hypercholesterolemia (FH) is a primary hyperlipemia. It is an autosomal dominant genetic disorder of lipoproteins metabolism mainly caused by mutations in the low density lipoprotein receptor gene (LDLR). We aimed to investigate the functional impact on the low density lipoprotein receptor (LDLR) activity of six uncharacterised variants located in the coding region of the LDLR gene, namely c.428G > T, c.640T > C, c.1708C > T, c.1736A > T, c.1981C > G and c.2114C > G (NM_000527.4) and to attempt to define their clinical status. MethodsFunctional studies were carried out using site-directed mutagenesis techniques and expression of LDLR protein in vitro. Results were correlated with clinical data and in silico analyses in order to assess the physiopathological role of these variants. ResultsThis work provides functional information about 6 uncharacterised mutations in LDLR. ConclusionsThe six variants studied here appeared to affect the LDLR function in vitro to different degrees, ranging from receptors with normal to slightly reduced activity to receptors exhibiting less than 10% of the wild-type activity. According to these studies and The American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines, two variants could be classified as “Likely Benign” (p.(Ala705Gly) and p.(Leu570Phe)), three variants as “Pathogenic” (p.(Asp579Val), p.(Cys143Phe) and p.(Trp214Arg)) and one variant as “Likely Pathogenic” (p.(Pro661Ala)).

Blank roasting and bioleaching of stone coal for vanadium recycling

Abstract The recovery and utilization of vanadium from stone coal is of great significance because it is an important metal. In the present study, a cleaner metal extraction method to recover vanadium from low-grade stone coal using a novel strain of Bacillus mucilaginosus (BM) and an enhanced bioleaching procedure was explored. The efficient leaching bacteria of BM-5070 were obtained by the atmospheric and room temperature plasma (ARTP)-hydroxylamine hydrochloride compound mutagenesis technique. The results show that BM-5070 had the highest V leaching rate. After a 20-day bioleaching period, the V leaching rate of BM-5070 was 5.5 percentage points higher than that of the original bacteria. Moreover, the pretreatment of stone coal by blank roasting without any additives can further promote the vanadium dissolution, and the V leaching rate reached 85% after bioleaching for 20 days, which was 57 percentage points higher than that of without roasting. This is due to the decrease in the content of V (III) and the increase in the V (V) and V (IV) content after blank roasting, which is beneficial to the leaching of vanadium. The bioleaching behavior was elucidated through the bacteria-mineral contact leaching and non-contact leaching test. The results show that the V leaching rate (81.0%) in the contact mode was much higher than that in the non-contact leaching mode (64.4%), indicating that the bioleaching of vanadium occurs with direct action and indirect action. The results of our study could significantly contribute to a green method for recovering vanadium from stone coal.

European Court of Justice delivers no justice to Europe on genome-edited crops.

European Court of Justice delivers no justice to Europe on genome-edited crops.

Statistical Analysis of the Benefits of Focused Saturation Mutagenesis in Directed Evolution Based on Reduced Amino Acid Alphabets

Directed evolution of stereo-, regio-, and chemoselective enzymes has enriched the toolbox of synthetic organic chemistry. Among the different gene mutagenesis techniques, saturation mutagenesis (S...

The EU regulatory framework on genetically modified organisms (GMOs).

The European Union (EU) legislation on genetically modified organisms (GMOs) aims to ensure a high level of protection for human, animal and environmental health and a well-functioning EU internal market. The framework regulates the release of GMOs into the environment and their use as, or in, food and feed. It has three main pillars: pre-market authorisation based on a prior risk assessment, traceability and labelling. Within this legal framework, the EU has authorised the placing on the market of 118 GMOs so far. These have been obtained through long-standing techniques of genetic modification, namely transgenesis. Following the adoption of the GMO legislation, new techniques of genetic modification, including new mutagenesis techniques, have been developed, which have raised questions regarding the applicability of the GMO legislation and attracted a lot of attention from stakeholders and the general public. This article provides an overview of EU GMO legislation and implementation of the EU Court of Justice ruling on organisms obtained by mutagenesis techniques, issued in July 2018. It also updates on the recent initiatives by the European Commission and EU Member States on new developments in biotechnology. The manuscript is based on the author's contribution at the OECD Conference on Genome Editing, Applications in Agriculture, Implications for Health, Environment and Regulation held in Paris on 28-29 June 2018. It is complemented with updated information.

What is the available evidence for the range of applications of genome-editing as a new tool for plant trait modification and the potential occurrence of associated off-target effects: a systematic map

BackgroundWithin the last decades, genome-editing techniques such as CRISPR/Cas, TALENs, Zinc-Finger Nucleases, Meganucleases, Oligonucleotide-Directed Mutagenesis and base editing have been developed enabling a precise modification of DNA sequences. Such techniques provide options for simple, time-saving and cost-effective applications compared to other breeding techniques and hence genome editing has already been promoted for a wide range of plant species. Although the application of genome-editing induces less unintended modifications (off-targets) in the genome compared to classical mutagenesis techniques, off-target effects are a prominent point of criticism as they are supposed to cause unintended effects, e.g. genomic instability or cell death. To address these aspects, this map aims to answer the following question: What is the available evidence for the range of applications of genome-editing as a new tool for plant trait modification and the potential occurrence of associated off-target effects? This primary question will be considered by two secondary questions: One aims to overview the market-oriented traits being modified by genome-editing in plants and the other explores the occurrence of off-target effects.MethodsA literature search in nine bibliographic databases, Google Scholar, and 47 web pages of companies and governmental agencies was conducted using predefined and tested search strings in English language. Articles were screened on title/abstract and full text level for relevance based on pre-defined inclusion criteria. The relevant information of included studies were mapped using a pre-defined data extraction strategy. Besides a descriptive summary of the relevant literature, a spreadsheet containing all extracted data is provided.ResultsAltogether, 555 relevant articles from journals, company web pages and web pages of governmental agencies were identified containing 1328 studies/applications of genome-editing in model plants and agricultural crops in the period January 1996 to May 2018. Most of the studies were conducted in China followed by the USA. Genome-editing was already applied in 68 different plants. Although most of the studies were basic research, 99 different market-oriented applications were identified in 28 different crops leading to plants with improved food and feed quality, agronomic value like growth characteristics or increased yield, tolerance to biotic and abiotic stress, herbicide tolerance or industrial benefits. 252 studies explored off-target effects. Most of the studies were conducted using CRISPR/Cas. Several studies firstly investigated whether sites in the genome show similarity to the target sequence and secondly analyzed these potential off-target sites by sequencing. In around 3% of the analyzed potential off-target sites, unintended mutations were detected. Only a few studies conducted off-target analyses using unbiased detection methods (e.g. whole genome sequencing). No off-target effects that could be correlated to the genome-editing process were identified in these studies.ConclusionsThe rapid adoption in plant breeding was demonstrated by a considerable number of market oriented applications (crops and traits) described in publications worldwide. Studies investigating off-target effects are very heterogeneous in their structure and design. Therefore, an in-depth assessment regarding their weight of evidence is mandatory.

Antitumor Potential of Fibulin-5 in Breast Cancer Cells Depends on Its RGD Cell Adhesion Motif.

Different components of the tumor microenvironment can be either tumor-promoting or tumor-suppressive agents depending on factors which are not fully understood. Fibulins are components of the extracellular matrix from different tissues and constitute a clear example of this dual function. In fact, fibulins may either support tumor growth or abolish progression of malignant cells depending on the crosstalk between tumor cells and their surrounding stroma through mechanisms that remain to be elucidated. Among all fibulins, fibulin-5 contains a particular structural hallmark which consists in the presence of a RGD motif within its architecture. Previous reports have highlighted the importance of the interaction of this motif with integrins, and not only in normal functions but also in a tumor context. Site-Directed Mutagenesis technique was employed to introduce the change RGD to RGE (RGD-to-RGE) within Fbln5 cDNA sequence. Cell proliferation was measured using the MTT assay or by counting Ki-67 positive cell nuclei. Cell adhesion was analysed using culture plates coated with different extracellular matrix components. Cell invasion was evaluated using 24-well Matrigel-coated invasion chambers, and mammosphere formation was monitored using ultralow attachment culture plates. BALB/c mice were employed to induce subcutaneous tumors. The RGD-to-RGE change alters the capacity of breast cancer cells to adhere to different extracellular matrix proteins as well as to αvβ3 and α5β1 integrins, and promotes protumor effects using different cell-based assays. Moreover, 4T1 cells, a mouse breast cancer cell line model, shows an increased capacity to generate tumors when exogenously expresses fibulin-5 with a RGD-to-RGE change, and such capacity is similar to that shown for 4T1 cells with an interfered Fbln5 gene. These data highlight the importance of the RGD motif of fibulin-5 to induce antitumor effects and provide new insights into the involvement of fibulins in tumor processes.

The Recent Progresses in Chemical Synthesis of Proteins with Site-Specific Lysine Post-translational Modifications

In the past two decades, a plethora of lysine (Lys) posttranslational modifications (PTMs) has been discovered on proteins, major groups are acylation, alkylation, and ubiquitination. Although considered biologically important, functional annotation of proteins with Lys PTMs has largely fallen behind the discovery. One grand challenge of characterizing proteins with PTMs is the procurement of homogenously modified proteins. To resolve this obstacle, sophisticated methods have been developed. These include total synthesis, semisynthesis that is based on native chemical ligation, expressed protein ligation, and enzyme-catalyzed peptide ligation, and the amber-suppression based noncanonical amino acid mutagenesis technique that may need to couple with follow-up bioorthogonal chemistry. This review summarizes currently identified significant PTMs and chemical biology methods for their installation in proteins. We hope that the current review will provide helpful insights and critical perspectives to this important research frontier.

Comparative study of alpha amylase producing mutated and unmutated Ba-cillus strains by taking cost effective measures

Bacillus species is considered as most appropriate bacterial specie for extracellular production of alpha amylase at commercial level. The alpha amylase is in high demand in a lot of food, beverage, and textile industries. In recent years, researchers have made many attempts to scale up the production of alpha amylase by using various strategies. The current study deals with the isolation, identification of Bacillus species and evaluation of their alpha amylase activity by random mutagenesis. Amylase producing bacteria were isolated from soil sample and further categorized via 16S rRNA technique. Random mutagenesis technique (MNNG and UV radiation) was employed to enhance the production of an enzyme. Mutants were then assessed for alpha amylase production via solid state fermentation using different agro-industrial waste, i.e. potato peels, apple peels, banana peels, wheat bran and rice husk. Different parameters such as incubation temperature, incubation period and pH were optimized to obtain maximum yield. Optimum amylase production (207.84U/ml) was obtained after 48 hours of incubation at 37°C by using MNNG mutant and potato peels. This study reveals that Bacillus cereus has the potential of alpha amylase production. It further suggests that secretion of alpha amylase can further be maximized by improving the strain genetically and optimizing the fermentation conditions. Keywords: Alpha amylase; Bacillus; MNNG; 16S rRNA; UV radiation http://dx.doi.org/10.19045/bspab.2019.80122

Expanding Avenue of Fast Neutron Mediated Mutagenesis for Crop Improvement.

Fast neutron (FN) radiation mediated mutagenesis is a unique approach among the several induced mutagenesis methods being used in plant science in terms of impacted mutations. The FN mutagenesis usually creates deletions from few bases to several million bases (Mb). A library of random deletion generated using FN mutagenesis lines can provide indispensable resources for the reverse genetic approaches. In this review, information from several efforts made using FN mutagenesis has been compiled to understand the type of induced mutations, frequency, and genetic stability. Concerns regarding the utilization of FN mutagenesis technique for a plant with different level of ploidy and genome complexity are discussed. We have highlighted the utility of next-generation sequencing techniques that can be efficiently utilized for the characterization of mutant lines as well as for the mapping of causal mutations. Pros and cons of mapping by mutation (MutMap), mutant chromosome sequencing (MutChromSeq), exon capture, whole genome sequencing, MutRen-Seq, and different tilling approaches that can be used for the detection of FN-induced mutation has also been discussed. Genomic resources developed using the FN mutagenesis have been catalogued wooing to meaningful utilization of the available resources. The information provided here will be helpful for the efficient exploration for the crop improvement programs and for better understanding of genetic regulations.

Derivation and maintenance of mouse haploid embryonic stem cells.

Ploidy represents the number of chromosome sets in a cell. Although gametes have a haploid genome (n), most mammalian cells have diploid genomes (2n). The diploid status of most cells correlates with the number of probable alleles for each autosomal gene and makes it difficult to target these genes via mutagenesis techniques. Here, we describe a 7-week protocol for the derivation of mouse haploid embryonic stem cells (hESCs) from female gametes that also outlines how to maintain the cells once derived. We detail additional procedures that can be used with cell lines obtained from the mouse Haplobank, a biobank of >100,000 individual mouse hESC lines with targeted mutations in 16,970 genes. hESCs can spontaneously diploidize and can be maintained in both haploid and diploid states. Mouse hESCs are genomically and karyotypically stable, are innately immortal and isogenic, and can be derived in an array of differentiated cell types; they are thus highly amenable to genetic screens and to defining molecular connectivity pathways.

Fitness of unregulated human Ras mutants modeled by implementing computational mutagenesis and machine learning techniques

Ras proteins play a pivotal role as oncogenes by participating in diverse signaling events, including those linked to cell growth, differentiation, and proliferation. Using experimental fitness data and implementing artificial intelligence and a computational mutagenesis technique, we developed models that reliably predict fitness for all single residue mutants of H-ras proto-oncogene protein p21. The computational mutagenesis generated a feature vector of protein structural changes for each variant, and these data correlated well with fitness. Random forest classification and tree regression machine learning algorithms were implemented for training predictive models. Cross-validations were used to evaluate model performance, and control experiments were performed to assess statistical significance. Classification models revealed a balanced accuracy rate as high as 82%, with a Matthew's correlation of 0.63, and an area under ROC curve of 0.90. Similarly, regression models displayed Pearson's correlation reaching 0.79. On the other hand, control data sets led to performance values consistent with random guessing. Comparisons with several related state-of-the-art methods reflected favorably on our trained models. This H-Ras proof-of-principle study suggests a complementary approach for understanding mechanisms with which other proteins are involved in oncogenesis, including related Ras isoforms, and for providing useful insights into designing future diagnostic and treatment modalities.

A novel CRISPR-directed gene editing method that catalyzes precise gene segment replacement in vitro enabling multiplex site-directed mutagenesis.

e14688 Background: Functional analysis of the multitude of mutations found in tumors is a major goal to better understand their role and to optimize patient treatment. PCR-based site-directed mutagenesis (SDM) techniques are often used to engineer these variants. While these tools are efficient, they are not without significant limitations, most notably off-site mutagenesis, limited scalability and lack of multiplexing capabilities. To overcome many of these limitations, we describe a novel, fast and simple method for the introduction of both simple and complex gene mutations in plasmid DNA by using in vitro CRISPR based DNA editing. Methods: For each mutation, a specifically designed pair of CRISPR/Cas12a ribonucleoprotein complexes are used to execute site-specific double-strand breaks on plasmid DNA enabling the excision of a defined DNA fragment. This is followed by donor DNA replacement and bacterial colony expansion. We term this method, CRISPR-directed DNA Mutagenesis (CDM). Results: Using CDM we have been able to synthesize known oncogenic mutations as well as novel variants in 8 different cancer genes. These mutations have been synthesized with over 60% success rate, compared to about 40% success rate in SDM. More importantly, we show that in the CDM method there were no off-site mutations eliminating the need to sequence large portions of the gene. Conclusions: We have developed a novel multiplex site-directed mutagenesis method that can generate multiple unique mutations simultaneously within plasmids. CDM has proven capable of precise, rapid and robust mutation synthesis, including single base point mutations, site-specific deletions, insertions and duplications within targeted plasmids.

TRPM4 and TRPM5 Channels Share Crucial Amino Acid Residues for Ca2+ Sensitivity but Not Significance of PI(4,5)P2.

Transient receptor potential melastatin member 4 (TRPM4) and 5 (TRPM5) channels are Ca2+-activated nonselective cation channels. Intracellular Ca2+ is the most important regulator for them to open, though PI(4,5)P2, a membrane phosphoinositide, has been reported to regulate their Ca2+-sensitivities. We previously reported that negatively-charged amino acid residues near and in the TRP domain are necessary for the normal Ca2+ sensitivity of TRPM4. More recently, a cryo-electron microscopy structure of Ca2+-bound (but closed) TRPM4 was reported, proposing a Ca2+-binding site within an intracellular cavity formed by S2 and S3. Here, we examined the functional effects of mutations of the amino acid residues related to the proposed Ca2+-binding site on TRPM4 and also TRPM5 using mutagenesis and patch clamp techniques. The mutations of the amino acid residues of TRPM4 and TRPM5 reduced their Ca2+-sensitivities in a similar way. On the other hand, intracellular applications of PI(4,5)P2 recovered Ca2+-sensitivity of desensitized TRPM4, but its effect on TRPM5 was negligible. From these results, the Ca2+-binding sites of TRPM4 and TRPM5 were shown to be formed by the same amino acid residues by functional analyses, but the impact of PI(4,5)P2 on the regulation of TRPM5 seemed to be smaller than that on the regulation of TRPM4.

Phenotypic and molecular characterisation of novel Vicia faba germplasm with tolerance to acetohydroxyacid synthase‐inhibiting herbicides (AHAS) developed through mutagenesis techniques

Faba bean (Vicia faba L.) is an important crop in Australian farming systems, however, weed control is a major constraint due to a lack of in-crop broadleaf herbicide options. To address this, we developed acetohydroxyacid synthase (AHAS) inhibitor herbicide tolerance in faba bean using mutagenesis techniques. Dose-response experiments, agronomic field evaluation and DNA sequencing of the AHAS gene were used to quantify and validate tolerance traits. Four M2 faba bean single-plant biotypes (IMI-1, IMI-2, IMI-3 and IMI-4) at a frequency of 3.63 × 10-6 were successfully recovered. Molecular characterisation of the AHAS gene identified two known target site mutations (resulting in protein substitutions Ala205Val and Ser653Asn) conferring tolerance. Phenotypic characterisation found that both mutations conferred high levels of tolerance to the imidazolinone herbicide imazapyr. However, although the Ala205Val substitution showed improved levels of cross-tolerance to a range of sulfonylurea chemistries, the Ser653Asn substitution did not. In the field, IMI-3 showed the highest level of agronomic tolerance across a range of imidazolinone herbicides. Mutagenesis techniques were successful in the development of tolerance to AHAS inhibitor herbicides in faba bean, and could facilitate the first safe in-crop broadleaf herbicide control option in Australian faba bean production. © 2019 Society of Chemical Industry.

Kub5‐Hera deficiency compromises homologous recombination and enhances vulnerability to PARP inhibition in BRCA‐proficient breast cancers

PurposeIdentification of novel factors that compromise homologous recombination (HR) is key toward innovative strategies to expand the use of poly(ADP‐ribose) polymerase (PARP) inhibitors beyond BRCA deficiency. Our lab discovered that partial loss of Kub5‐Hera (K‐H), a transcription termination factor, stimulates the formation of various BRCA‐related cancers in female mice that was accelerated by single exposure to ionizing radiation (IR, 1 Gy). Here, we investigated how K‐H functionally impacts homologous recombination (HR) and cellular response to PARP inhibition in a subset of BRCA‐proficient breast cancer cell linesExperimental MethodsThe functional impact of K‐H alterations in a subset of breast cancer cell lines were assessed by targeted gene silencing, bioinformatics, mutagenesis techniques, plasmid reporter assays, immunofluorescence, microarray/qPCR analyses, chromatin immunoprecipitation, colony formation assays, rescue experiments and Western blots.ResultsKub5‐Hera promotes the expression of a critical DNA damage response and repair effector, cyclin dependent kinase 1 (CDK1), which phosphorylates BRCA1 for efficient repair of double‐strand breaks (DSBs). Mechanistically, we demonstrate that K‐H binds RNA Polymerase II (RNAPII) at the CDK1 promoter to efficiently enhance CDK1 transcription. K‐H depletion compromises HR, but upregulates poly(ADP‐ribose) polymerase 1 (PARP1) activity for cancer cell survival. Genetic and pharmacological ablation of PARP1 activity in a subset of BRCA‐proficient cell lines with aberrant K‐H reduction leads to synthetic lethality, which is reversed by re‐expression of wild‐type K‐H but not the mutant with reduced RNAPII binding.ConclusionsK‐H deficiency promotes genomic instability and a state of “BRCAness” in a subset of breast cancers. These cells may have an exploitable addiction to PARP‐mediated DNA repair for survival that could greatly expand the use of PARP inhibitors beyond BRCA mutations. Collectively, these results suggest that aberrant K‐H alterations may impact overall cellular response and survival to DNA damage and have clinical implications in cancer.Support or Funding InformationNIH R01 CA210489 (PI: David A. Boothman); Minority Supplement CURE Award CA139217‐05S1 and T32CA124334 to E.A. MoteaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Adenomatous Polyposis Coli‐like protein (APCLP) Functions as a Novel Negative Regulator of NF‐kB Signaling in Colon Cancer Cells

Colon cancer (CRC) is the second leading cause of cancer related deaths in the United States. CRC is marked by aberrantly activated signaling of the nuclear factor of kappaB (NF‐kB), a family of transcription factors which regulate wide varieties of cellular processes. Despite recent advances in comprehension of players of NF‐kB signaling, deeper understanding of its regulation is imperative for the development of novel cancer therapeutics. Using the powerful validation‐based insertional mutagenesis (VBIM) technique, we recently discovered APCLP as a novel negative regulator of NF‐kB. The objective of this study is to elucidate the role APCLP in regulating NF‐kB signaling in CRC at the molecular and biological levels and to understand the mechanism by which this regulation occurs. To determine the biological effect of APCLP on NF‐kB signaling, we used lentiviral vectors to either overexpress or knockdown (shRNA) APCLP in human CRC cell lines (HT‐29, HCT116, DLD‐1). We show that overexpression of APCLP decreased the NF‐kB activity, reduced cellular proliferation, migratory ability, as well as anchorage‐independent growth of cells while knockdown of APCLP had an inverse effect. Furthermore, in vivo experiments in a xenograft mouse model confirmed that APCLP overexpression impeded whereas shRNA knockdown promoted tumor growth. To study the mechanism by which APCLP regulated NF‐kB signaling, we conducted co‐Immunoprecipitation experiments and confirmed that APCLP and the major subunit of NF‐kB, p65, may complex or bind directly to each other. Studies are ongoing regarding the mechanism of interaction between APCLP and p65. In summary, discovery of APCLP and understanding of its molecular mechanism and biological function are significant because the knowledge acquired from this study could lead to utilization of APCLP as a potential biomarker and therapeutic target in CRC as well as other cancers that are driven by hyperactivated NF‐kB.Support or Funding InformationThis work is supported by NIH‐NIGMS Grant (# 1R01GM120156‐01A1 to TL), NIH‐NCI Grant (# 1R03 CA223906‐01 to TL), V foundation Kay Yow Cancer Fund (Grant 4486242 to TL), and 100 VOH Grant (# 2987613 to TL).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CRISPR-Directed Gene Editing Catalyzes Precise Gene Segment Replacement In Vitro Enabling a Novel Method for Multiplex Site-Directed Mutagenesis.

Much of our understanding of eukaryotic genes function comes from studies of the activity of their mutated forms or allelic variability. Mutations have helped elucidate how members of an intricate pathway function in relation to each other and how they operate in the context of the regulatory circuitry that surrounds them. A PCR-based site-directed mutagenesis technique is often used to engineer these variants. While these tools are efficient, they are not without significant limitations, most notably off-site mutagenesis, limited scalability, and lack of multiplexing capabilities. To overcome many of these limitations, we now describe a novel method for the introduction of both simple and complex gene mutations in plasmid DNA by using in vitro DNA editing. A specifically designed pair of CRISPR-Cas12a ribonucleoprotein complexes are used to execute site-specific double-strand breaks on plasmid DNA, enabling the excision of a defined DNA fragment. Donor DNA replacement is catalyzed by a mammalian cell-free extract through microhomology annealing of short regions of single-stranded DNA complementarity; we term this method CRISPR-directed DNA mutagenesis (CDM). The products of CDM are plasmids bearing precise donor fragments with specific modifications and CDM could be used for mutagenesis in larger constructs such as Bacterial Artificial Chromosome (BACs) or Yeast Artificial Chromosome (YACs). We further show that this reaction can be multiplexed so that product molecules with multiple site-specific mutations and site-specific deletions can be generated in the same in vitro reaction mixture. Importantly, the CDM method produces fewer unintended mutations in the target gene as compared to the standard site-directed mutagenesis assay; CDM produces no unintended mutations throughout the plasmid backbone. Lastly, this system recapitulates the multitude of reactions that take place during CRISPR-directed gene editing in mammalian cells and affords the opportunity to study the mechanism of action of CRISPR-directed gene editing in mammalian cells by visualizing a multitude of genetic products.

A Counterselectable Sucrose Sensitivity Marker Permits Efficient and Flexible Mutagenesis in Streptococcus agalactiae.

Streptococcus agalactiae (group B Streptococcus [GBS]) is a cause of severe infections, particularly during the newborn period. While methods exist for generating chromosomal mutations in GBS, they are cumbersome and inefficient and present significant challenges if the goal is to study subtle mutations, such as single-base-pair polymorphisms. To address this problem, we have developed an efficient and flexible GBS mutagenesis protocol based on sucrose counterselection against levansucrase (SacB) expressed from a temperature-selective shuttle vector. GBS containing the SacB expression cassette demonstrates lethal sensitivity to supplemental sucrose whether the plasmid DNA is replicating outside of the chromosome or has been integrated during a crossover event. Transmission electron microscopy shows that SacB-mediated lethal sucrose sensitivity results from the accumulation of inclusion bodies that eventually lead to complete degradation of normal cellular architecture and subsequent lysis. We used this new mutagenesis technique to generate an in-frame, allelic exchange knockout of the GBS sortase gene srtA, demonstrating that >99% of colonies that emerge from our protocol had the expected knockout phenotype and that among a subset tested by sequencing, 100% had the correct genotype. We also generated barcoded nonsense mutations in the cylE gene in two GBS strains, showing that the approach can be used to make small, precise chromosomal mutations.IMPORTANCE The ability to generate chromosomal mutations is fundamental to microbiology. Historically, however, GBS pathogenesis research has been made challenging by the relative genetic intractability of the organism. Generating a single knockout in GBS using traditional techniques can take many months, with highly variable success rates. Furthermore, traditional methods do not offer a straightforward way to generate single-base-pair polymorphisms or other subtle changes, especially to noncoding regions of the chromosome. We have developed a new sucrose counterselection-based method that permits rapid, efficient, and flexible GBS mutagenesis. Our technique requires no additional equipment beyond what is needed for traditional approaches. We believe that it will catalyze rapid advances in GBS genetics research by significantly easing the path to generating mutants.