Genes Of Interest Research Articles

CuBe: a geminivirus-based copper-regulated expression system suitable for post-harvest activation.

The growing demand for sustainable platforms for biomolecule manufacturing has fuelled the development of plant-based production systems. Agroinfiltration, the current industry standard, offers several advantages but faces limitations for large-scale production due to high operational costs and batch-to-batch variability. Alternatively, here, we describe the CuBe system, a novel bean yellow dwarf virus (BeYDV)-derived conditional replicative expression platform stably transformed in Nicotiana benthamiana and activated by copper sulphate (CuSO4), an inexpensive and widely used agricultural input. The CuBe system utilizes a synthetic circuit of four genetic modules integrated into the plant genome: (i) a replicative vector harbouring the gene of interest (GOI) flanked by cis-acting elements for geminiviral replication and novelly arranged to enable transgene transcription exclusively upon formation of the circular replicon, (ii) copper-inducible Rep/RepA proteins essential for replicon formation, (iii) the yeast-derived CUP2-Gal4 copper-responsive transcriptional activator for Rep/RepA expression, and (iv) a copper-inducible Flp recombinase to minimize basal Rep/RepA expression. CuSO4 application triggers the activation of the system, leading to the formation of extrachromosomal replicons, expression of the GOI, and accumulation of the desired recombinant protein. We demonstrate the functionality of the CuBe system in N. benthamiana plants expressing high levels of eGFP and an anti-SARS-CoV-2 antibody upon copper treatment. Notably, the system is functional in post-harvest applications, a strategy with high potential impact for large-scale biomanufacturing. This work presents the CuBe system as a promising alternative to agroinfiltration for cost-effective and scalable production of recombinant proteins in plants.

Site-Specific Integration by Circular Donor Improves CRISPR/Cas9-Mediated Homologous Recombination in Human Cell Lines

The technology for obtaining the high-efficiency expression of target proteins through site-specific recombination has made progress. However, using the CRISPR/Cas9 system for site-specific integration of long fragments and the expression of active proteins remains a challenge. This study optimized the linear DNA circularization system, eliminated the prokaryotic plasmid backbone on the traditional foreign gene vector, and generated a homologous arm-free circular donor template with a single guide RNA target site (sgRNA TS). This strategy significantly increased the co-transfection efficiency of the 1.6 kb template and Cas9 plasmid by 1.15-fold, and the average knock-in (KI) efficiency of the 4.7 kb long-fragment template for the two target gene sites increased by 1.3-fold. Subsequently, we used rhBCHE as a reporter gene to efficiently integrate the 5.4 kb fragment containing the gene of interest (GOI) into specific sites in the HEK293T cell line to detect the expression of the circular template at different target sites. Overall, this study further verifies that the length of the circular donor is more conducive to non-homologous integration, and more importantly, we provide a simple and optimized strategy for the construction of long-fragment site integration cell lines.

Competition between homologous chromosomal DNA and exogenous donor DNA to repair CRISPR/Cas9-induced double-strand breaks in Aspergillus niger

BackgroundAspergillus niger is well-known for its high protein secretion capacity and therefore an important cell factory for homologous and heterologous protein production. The use of a strong promoter and multiple gene copies are commonly used strategies to increase the gene expression and protein production of the gene of interest (GOI). We recently presented a two-step CRISPR/Cas9-mediated approach in which glucoamylase (glaA) landing sites (GLSs) are introduced at predetermined sites in the genome (step 1), which are subsequently filled with copies of the GOI (step 2) to achieve high expression of the GOI.ResultsHere we show that in a ku70 defective A. niger strain (Δku70), thereby excluding non-homologous end joining (NHEJ) as a mechanism to repair double-stranded DNA breaks (DSBs), the chromosomal glaA locus or homologous GLSs can be used to repair Cas9-induced DSBs, thereby competing with the integration of the donor DNA containing the GOI. In the absence of exogenously added donor DNA, the DSBs are repaired with homologous chromosomal DNA located on other chromosomes (inter-chromosomal repair) or, with higher efficiency, by a homologous DNA fragment located on the same chromosome (intra-chromosomal repair). Single copy inter-chromosomal homology-based DNA repair was found to occur in 13–20% of the transformants while 80–87% of the transformants were repaired by exogenously added donor DNA. The efficiency of chromosomal repair was dependent on the copy number of the potential donor DNA sequences in the genome. The presence of five homologous DNA sequences, resulted in an increased number (35–61%) of the transformants repaired by chromosomal DNA. The efficiency of intra-chromosomal homology based DSB repair in the absence of donor DNA was found to be highly preferred (85–90%) over inter-chromosomal repair. Intra-chromosomal repair was also found to be the preferred way of DNA repair in the presence of donor DNA and was found to be locus-dependent.ConclusionThe awareness that homologous chromosomal DNA repair can compete with donor DNA to repair DSB and thereby affecting the efficiency of multicopy strain construction using CRISPR/Cas9-mediated genome editing is an important consideration to take into account in industrial strain design.

Functional Characterization of 11 Tentative Microneme Proteins in Type I RH Strain of Toxoplasma gondii Using the CRISPR-Cas9 System.

Toxoplasma gondii, a pathogenic apicomplexan parasite, infects approximately one third of the world's population and poses a serious threat to global public health. Microneme proteins (MICs) secreted by the microneme, an apical secretory organelle of T. gondii, play important roles in the invasion, motility, and intracellular survival of T. gondii. In this study, we selected 11 genes of interest (GOIs) of T. gondii, tentative MICs predicted to be localized in micronemes, and we used the CRISPR-Cas9 system to construct epitope tagging strains and gene knockout strains to explore the localization and function of these 11 tentative MICs. Immunofluorescence assay showed that nine tentative MICs (TGME49_243930, TGME49_200270, TGME49_273320, TGME49_287040, TGME49_261710, TGME49_205680, TGME49_304490, TGME49_245485, and TGME49_224620) were localized or partially localized in the microneme, consistent with the prediction. However, TGME49_272380 and TGME49_243790 showed different localizations from the prediction, being localized in the endoplasmic reticulum and the dense granule, respectively. Further functional characterization of the 11 RHΔGOI strains revealed that deletion of these 11 GOIs had no significant effect on plaque formation, intracellular replication, egress, invasion ability, and virulence of T. gondii. Although these 11 GOIs are not essential genes for the growth and virulence of tachyzoites of type I RH strain, they may have potential roles in other developmental stages or other genotypes of T. gondii. Thus, further research should be performed to explore the possible role of the nine mics and the other two GOIs in other life cycle stages and other genotypes of T. gondii.

Efficient circularization of protein-encoding RNAs via a novel cis-splicing system.

Circular RNAs (circRNAs) have emerged as a promising alternative to linear mRNA, owing to their unique properties and potential therapeutic applications, driving the development of novel approaches for their production. This study introduces a cis-splicing system that efficiently produces circRNAs by incorporating a ribozyme core at one end of the precursor, thereby eliminating the need for additional spacer elements between the ribozyme and the gene of interest (GOI). In this cis-splicing system, sequences resembling homologous arms at both ends of the precursor are crucial for forming the P9.0 duplex, which in turn facilitates effective self-splicing and circularization. We demonstrate that the precise recognition of the second transesterification site depends more on the structural characteristics of P9.0 adjacent to the ωG position than on the nucleotide composition of the P9.0-ωG itself. Further optimization of structural elements, like P10 and P1-ex, significantly improves circularization efficiency. The circRNAs generated through the cis-splicing system exhibit prolonged protein expression and minimal activation of the innate immune response. This study provides a comprehensive exploration of circRNA generation via a novel strategy and offers valuable insights into the structural engineering of RNA, paving the way for future advancements in circRNA-based applications.

MYO5B and the Polygenic Landscape of Very Early-Onset Inflammatory Bowel Disease in an Ethnically Diverse Population.

Genetic discovery in very early-onset inflammatory bowel disease (VEO-IBD) can elucidate not only the origins of VEO-IBD, but also later-onset inflammatory bowel disease. We aimed to investigate the polygenic origins of VEO-IBD in a cohort with a high proportion of Hispanic patients. Patients with VEO-IBD who underwent whole exome sequencing at our center were included. Genes were categorized as genes of interest (GOIs) (129 genes previously described to be associated with VEO-IBD) or non-GOIs. VEO-IBD "susceptibility" single nucleotide variants (SNVs) were identified through enrichment compared with gnomAD (Genome Aggregation Database) and ALFA (Allele Frequency Aggregator) and were scored by Combined Annotation Dependent Depletion for deleteriousness. Gene networks carrying susceptibility SNVs were created. Myosin 5b immunofluorescence was also studied. Fifty-six patients met inclusion criteria, and 32.1% identified as Hispanic. Monogenic disease was infrequent (8.9%). Significant enrichment of GOI susceptibility SNVs was observed, notably in MYO5B, especially in Hispanics. MEFV, TNFAIP3, SH3TC2, and NCF2 were also central participants in the GOI networks. Myosin 5b immunofluorescence in colonic mucosa was significantly reduced in those with MYO5B susceptibility SNVs compared with control subjects. Seven genes (ESRRA, HLA-DQ1, RETSAT, PABPC1, PARP4, CCDC102A, and SUSD2) were central participants in the non-GOI networks. Our results support the polygenic nature of VEO-IBD, in which key participants, like MYO5B, were identified through network analytics. Rare variant load within susceptibility genes may be relevant not only for the genetic origins of inflammatory bowel disease, but also for the age of disease onset. Our findings could guide future work in precision medicine.

Organ-Specific Gene Expression Control Using DNA Origami-Based Nanodevices.

Nanodevices that function in specific organs or cells are one of the ultimate goals of synthetic biology. The recent progress in DNA nanotechnology such as DNA origami has allowed us to construct nanodevices to deliver a payload (e.g., drug) to the tumor. However, delivery to specific organs remains difficult due to the fragility of the DNA nanostructure and the low targeting capability of the DNA nanostructure. Here, we constructed tough DNA origami that allowed us to encapsulate the DNA origami into lipid-based nanoparticles (LNPs) under harsh conditions (low pH), harnessing organ-specific delivery of the gene of interest (GOI). We found that DNA origami-encapsulated LNPs can increase the functionality of payload GOIs (mRNA and siRNA) inside mouse organs through the contribution from different LNP structures revealed by cryogenic electron microscope (Cryo-EM). These data should be the basis for future organ-specific gene expression control using DNA origami nanodevices.

Somatic tissue testing in prostate cancer using the Oncomine V3 panel: A single-institution retrospective analysis.

e17002 Background: Approximately 20-30% of prostate cancer (PC) patients have a mutation in the homologous recombination repair or mismatch repair genes, 50% of which are somatic. These mutations have prognostic and predictive implications. Somatic tissue testing (STT) was implemented at Sunnybrook Health Sciences Centre using the Oncomine V3 panel, which includes 161 genes, but only four genes (BRCA1/2, ATM, PALB2) are reported due to funding restrictions. We describe our centre’s testing experience and the utility of reporting beyond these four genes. Methods: Sixty-two PC patients between May 1, 2021 - January 31, 2023 were included. Clinical characteristics were gathered via chart review. Somatic variants of genes of interest (GOI) within the Oncomine V3 panel were extracted by an annotation specialist. Results: STT was ordered at diagnosis in 69.4% of cases (n=42) vs. at progression in 27.4% (n=17), 3 cases were external and lacked clinical context. Tests were ordered in the locoregional disease setting in 46.8% (n=29), 37.1% (n=23) in the metastatic castrate-sensitive setting, 8.1% (n=5) in the metastatic castrate-resistant (CR) setting and 3.2% (n=2) in the non-metastatic CR setting. Tests were ordered by Urology or Pathology (reflex testing) in 64.5% (n=40), by Medical Oncology in 16% (n=10), by Radiation Oncology in 13% (n=8) and by others in 6.5% (n=4). Fifty-one samples (82.2%) were of the prostate and 11 (17.7%) were of metastatic sites. The median sample age was 8 days (range 0-5781). The median test turn-around time was 28.5 days (range 5-57). There were 378 variants within the GOI and meeting the limit of detection [1] , of which 63 (16.7%) were tier 1 or 2 and are listed by gene in the table. Eight of 63 variants (12.7%) were within the four funded genes whereas 55/63 (87.3%) were within unfunded genes of clinical significance. CDK12, FANCA, NBN (n=7, 11.2%) are investigated in poly (ADP-ribose) polymerase inhibitor trials and PMS2 deficiency (n=1, 1.6%) can select for immune checkpoint inhibitors. PIK3R1/PIK3CA/AKT1-mutated tumors (n=11, 17.7%) have shown response to AKT-inhibitors. SPOP-mutated tumors (n=6, 9.7%) are associated with prolonged response to androgen receptor blockade whereas PTEN/TP53/RB1-negative tumors (n=12, 19.4%) are associated with anti-androgen resistance and worse prognosis. Conclusions: The majority of STT variants identified were beyond the currently funded genes in Ontario, and many have important prognostic and predictive implications. This highlights that STT early in the disease course using expanded gene panels may help in delivering personalized PC care. [Table: see text]

Development of a telomere vector-based approach to overcome limitations caused by lethal phenotypes in the study of essential genes in Magnaporthe oryzae.

Reverse genetic approaches are common tools in genomics for elucidating gene functions, involving techniques such as gene deletion followed by screening for aberrant phenotypes. If the generation of gene deletion mutants fails, the question arises whether the failure stems from technical issues or because the gene of interest (GOI) is essential, meaning that the deletion causes lethality. In this report, we introduce a novel method for assessing gene essentiality using the phytopathogenic ascomycete Magnaporthe oryzae. The method is based on the observation that telomere vectors are lost in transformants during cultivation without selection pressure. We tested the hypothesis that essential genes can be identified in deletion mutants co-transformed with a telomere vector. The M. oryzae gene MoPKC, described in literature as essential, was chosen as GOI. Using CRISPR/Cas9 technology transformants with deleted GOI were generated and backed up by a telomere vector carrying a copy of the GOI and conferring fenhexamid resistance. Transformants in which the GOI deletion in the genome was not successful lost the telomere vector on media without fenhexamid. In contrast, transformants with confirmed GOI deletion retained the telomere vector even in absence of fenhexamid selection. In the latter case, the maintenance of the telomere indicates that the GOI is essential for the surveillance of the fungi, as it would have been lost otherwise. The method presented here allows to test for essentiality of genes when no mutants can be obtained from gene deletion approaches, thereby expanding the toolbox for studying gene function in ascomycetes.

Synergistic promotion of transient transgene expression in CHO cells by PDI/XBP-1s co-transfection and mild hypothermia.

Transient gene expression system is an important tool for rapid production of recombinant proteins in Chinese hamster ovary (CHO) cells. However, their low productivity is the main hurdle to overcome. An effective approach through which to obtain high protein yield involves targeting transcriptional, post-transcriptional events (PTEs), and culture conditions. Here, we investigated the effects of protein disulfide isomerase (PDI) and spliced X-box binding protein 1 (XBP-1s) co-overexpression combined with mild hypothermia on the transient yields of recombinant proteins in CHO cells. The results showed that the gene of interest (GOI) and the PDI/XBP-1s helper vector at a co-transfection ratio of 10:1 could obviously increase transient expression level of recombinant protein in CHO cells. However, PDI/XBP-1s overexpression had no significance effect on the mRNA levels of the recombinant protein, suggesting that it targeted PTEs. Moreover, the increased production was due to the enhancing of cell specific productivity, not related to cell growth, viability, and cell cycle. In addition, combined PDI/XBP-1s co-overexpression and mild hypothermia could further improve Adalimumab expression, compared to the control/37°C and PDI/XBP-1s/37°C, the Adalimumab volume yield of PDI/XBP-1s/33°C increased by 203% and 142%, respectively. Mild hypothermia resulted in 3.52- and 2.33-fold increase in the relative mRNA levels of PDI and XBP-1s, respectively. In conclusion, the combination of PDI/XBP-1s overexpression and culture temperature optimization can achieve higher transient expression of recombinant protein, which provides a synergetic strategy to improve transient production of recombinant protein in CHO cells.

Multi-omics kinetic analysis of recombinant adeno-associated virus production by plasmid transfection of HEK293 cells.

Recombinant adeno-associated virus (rAAV) is among the most commonly used vectors for gene therapy. It is commonly produced by transfection of HEK293 cells with three plasmids each containing the vector genome including gene of interest (GOI), helper functions, and rep and cap genes for genome replication and capsid formation. To meet the potential clinical needs, the productivity of the production system needs to be enhanced. A better process characterization of the production system will further advance our insights into ways to enhance productivity. Here, we employed transcriptomic analysis to quantify the dynamics of different isoforms of viral transcripts and to assess the shift of cellular physiology, and deployed targeted proteomic analysis for absolute quantification of viral proteins and tandem mass tags (TMTs) for assessing cellular responses at the protein level. Functional analysis at transcriptome and proteome levels identified defense and immune response, unfolded protein response, p53 signaling as enriched. The small molecule additive intervention study based on functional analysis showed the potential of such omics-guided productivity enhancement. Together, multi-omics analysis advanced understanding of rAAV production and provided insight into enhancing rAAV production by plasmid transfection.

Synergistic and antagonistic interactions of oxybenzone and ocean acidification: new insight into vulnerable cellular processes in non-calcifying anthozoans.

Cnidarians face significant threats from ocean acidification (OA) and anthropogenic pollutants such as oxybenzone (BP-3). The convergence of threats from multiple stressors is an important area to investigate because of potential significant synergistic or antagonistic interactions. Real-time quantitative PCR was performed to characterize the expression profiles of twenty-two genes of interest (GOI) in sea anemones (Exaiptasia diaphana) exposed to one of four treatments: 1) 96h of OA conditions followed by a 4h exposure to 20ppb BP-3; 2) Exposure to 4h 20ppb BP-3 without 96h of OA; 3) Exposure to 96h of OA alone; or 4) laboratory conditions with no exposure to BP-3 and/or OA. These 22 GOIs represent cellular processes associated with proton-dependent transport, sodium-dependent transport, metal cation binding/transport, extracellular matrix, amino acid metabolism/transport, immunity, and/or steroidogenesis. These 22 GOIs provide new insight into vulnerable cellular processes in non-calcifying anthozoans exposed to OA and BP-3. Expression profiles were categorized as synergistic, antagonistic, or additive of BP-3 in the presence of OA. Two GOIs were synergistic. Fifteen GOIs were antagonistic and the remaining five GOIs were additive in response to BP-3 in acidified seawater. A subset of these GOIs appear to be candidate biomarkers for future in situ investigations. In human health, proton-dependent monocarboxylate transporters (MCTs) are promising pharmacological targets and recognized as potential biomarkers. By comparison, these same MCTs appear to be targets of xenobiotic chemical pollutants in cnidarian physiology. In the presence of BP-3, a network of collagen synthesis genes are upregulated and antagonistic in their expression profiles. Cytochrome b561 is a critical protein required for collagen synthesis and in silico modeling demonstrates BP-3 binds in the pocket of cytochrome b561. Understanding the underlying molecular mechanisms of "drug-like" compounds such as BP-3 may lead to a more comprehensive interpretation of transcriptional expression profiles. The collective antagonistic responses of GOIs associated with collagen synthesis strongly suggests these GOIs should be considered candidate biomarkers of effect. GOIs with synergistic and additive responses represent candidate biomarkers of exposure. Results show the effects of OA and BP-3 are interactive with respect to their impact on cnidarians. This investigation offers mechanistic data that supports the expression profiles and underpins higher order physiological responses.

Combining a Base Deaminase Mutator with Phage-Assisted Evolution.

Phage-assisted evolution has emerged as a powerful technique for improving a protein's function by using mutagenesis and selective pressure. However, mutations typically occur throughout the host's genome and are not limited to the gene-of-interest (GOI): these undesirable genomic mutations can yield host cells that circumvent the system's selective pressure. Our system targets mutations specifically toward the GOI by combining T7 targeted mutagenesis and phage-assisted evolution. This system improves the structure and function of proteins by accumulating favorable mutations that can change its binding affinity, specificity, and activity.

Generation of Connexin-Expressing Stable Cell Pools.

Stable cell pools have the advantage of providing a definite, consistent, and reproducible transmission of a transgene of interest, compared to transient expression from a plasmid transfection. Stably expressing a transgene of interest in cells under induction is a powerful way to (switch on and) study a gene function in both in vitro and in vivo assays. Taking advantage of the ability of lentivirus (LV) to promote transgene delivery, and genomic integration and expression in both dividing and nondividing cells, a doxycycline-inducible transfer vector expressing a bicistronic transgene was developed to study the function of connexins in HeLa DH cells. Here, delving on connexin 32 (Cx32), we report how to use the backbone of this vector as a tool to generate stable pools to study the function of a gene of interest (GOI), especially with assays involving Ca2+ imaging, employing the GCaMP6s indicator. We describe a step-by-step protocol to produce the LV particle by transient transfection and the direct use of the harvested LV stock to generate stable cell pools. We further present step-by-step immunolabeling protocols to characterize the transgene protein expression by confocal microscopy using an antibody that targets an extracellular domain epitope of Cx32 in living cells, and in fixed permeabilized cells using high affinity anti-Cx32 antibodies. Using common molecular biology laboratory techniques, this protocol can be adapted to generate stable pools expressing any transgene of interest, for both in vitro and in vivo functional assays, including molecular, immune, and optical assays.

A highly efficient transposon vector system for recombinant protein expression in CHO cells

Chinese hamster ovary (CHO) cells are the most widely used mammalian host cells for the production of recombinant therapeutic proteins. Expression vectors are a key factor in determining the expression level of gene of interest (GOI) in CHO cells. Here, two transposon vector systems were constructed by adding two different pairs of inverted terminal repeats (ITR-1 and ITR-2), and four proteins of interest (POI), including enhanced green fluorescence protein (eGFP), secreted alkaline phosphatase (SEAP), human serum albumin (HSA) and Adalimumab (Adm) were expressed in CHO cells. The stably transfected cell pools and single cell clones were screened, and the expression levels of POIs were detected by qPCR, Western blot and ELISA. The results showed that both transposon vectors could improve the expression levels of POI at both mRNA and protein levels, while the ITR-2 sequence was more effective. Moreover, the positive cell clone rate of transposon system was higher than that of the control. In conclusion, the ITR-2 transposon vector system can be effectively used for the expression of recombinant proteins in CHO cells.

Abstract P2160: High-throughput Imaging Of Cardiomyocytes Using Biomimetic Tools Help Identify Therapeutic Targets

Cardiovascular disease is a leading cause of death and continues to require novel forms of therapies. Mutations in sarcomeres, the basic contractile units of cardiac muscle, causing disarray and loss of function are causal in many genetic cardiomyopathies. Here, we model sarcomeric disarray in vitro by culturing human induced pluripotent stem cell-derived cardiomyocytes (human iPSC-CMs) on tissue culture plates with biomimetic patterned grooves designed to simulate extracellular matrix. This culture system models the natural structure and phenotypic development of human physiology. Next, we created an Adeno-associated virus open reading frame (AAV-ORF) library of ~100 heart failure associated genes of interest (GOI) identified using publicly available human genetic data. We developed a high throughput screening (HTS) process to test the effects of overexpression of these GOIs on sarcomere morphology. Images of human iPSC-CMs treated with overexpression libraries were run through a custom MATLAB HTS image processing script named ‘Tamarack’, developed for quantifying human iPSC-CM sarcomere morphology. Tamarack enables quantification of sarcomere count, length, and orientation. Analysis of sarcomeres from the Tamarack code in the biomimetic plates allows for enhanced quantification of sarcomere structures, in particular, alignment, a metric missing from standard 2D culture plates. We found that treating cells with siRBM20 induced a sarcomere misalignment phenotype which we looked to rescue with overexpression of our GOI. Genes that increased alignment in an RBM20 background were considered top hits and moved to further analysis.

Genetic drivers in the natural history of chronic lymphocytic leukemia development as early as 16 years before diagnosis

Chronic lymphocytic leukemia (CLL) is preceded by monoclonal B-cell lymphocytosis (MBL), a potential CLL precursor state which can be detected in up to 17% in aged individuals. Recently, we described significant B-cell receptor immunoglobulin heavy chain (BCR IGH) gene repertoire skewing and clonotypic evolution up to 22 years before CLL diagnosis. However, pathobiological drivers during the earliest stages of MBL development remain incompletely characterized. In this study, we utilized the EuroClonality-NDC panel to sequence recurrently mutated genes in CLL in 39 peripheral blood samples from 16 CLL patients sampled up to 16 years prior to diagnosis. CLL diagnosis ranged from 5 months to 16 years after first blood sampling. Of 16 CLL patients, 8 (50%) presented with variants of interest in genes recurrently mutated in CLL such as NOTCH1, ATM, and SF3B1 . ATM variants and the IGLV3-21R110 mutation were present from the early stages of (pre)MBL development, while NOTCH1, SF3B1, and XPO1 variants arose closer to diagnosis. We additionally detected variants in FAT1 and PLCG2 as early as 10 years prior to CLL diagnosis. Overall, our data shows specific genetic drivers of CLL are associated with early and late stages of CLL development.

Enhancing Maize Transformation and Targeted Mutagenesis through the Assistance of Non-Integrating Wus2 Vector.

Efficient genetic transformation is a prerequisite for rapid gene functional analyses and crop trait improvements. We recently demonstrated that new T-DNA binary vectors with NptII/G418 selection and a compatible helper plasmid can efficiently transform maize inbred B104 using our rapid Agrobacterium-mediated transformation method. In this work, we implemented the non-integrating Wuschel2 (Wus2) T-DNA vector method for Agrobacterium-mediated B104 transformation and tested its potential for recalcitrant inbred B73 transformation and gene editing. The non-integrating Wus2 (NIW) T-DNA vector-assisted transformation method uses two Agrobacterium strains: one carrying a gene-of-interest (GOI) construct and the other providing an NIW construct. To monitor Wus2 co-integration into the maize genome, we combined the maize Wus2 expression cassette driven by a strong constitutive promoter with a new visible marker RUBY, which produces the purple pigment betalain. As a GOI construct, we used a previously tested CRISPR-Cas9 construct pKL2359 for Glossy2 gene mutagenesis. When both GOI and NIW constructs were delivered by LBA4404Thy- strain, B104 transformation frequency was significantly enhanced by about two-fold (10% vs. 18.8%). Importantly, we were able to transform a recalcitrant inbred B73 using the NIW-assisted transformation method and obtained three transgene-free edited plants by omitting the selection agent G418. These results suggest that NIW-assisted transformation can improve maize B104 transformation frequency and provide a novel option for CRISPR technology for transgene-free genome editing.

Real-Time Quantitative PCR: Primer Design, Reference Gene Selection, Calculations and Statistics.

Real-time quantitative PCR is a technique that can measure the content of the target nucleic acid sequence of interest in a given sample. It is mainly divided into absolute and relative quantitative methods. The relative quantification is mainly used in gene expressions for functional genomic and transcriptome studies. However, to use this technology accurately, there are some key points to master. First, specific primers need to be designed to ensure amplification of the gene of interest (GOI). Second, the appropriate reference gene or reference gene combination has to be selected. Finally, scientific gene expression level calculations and statistics are required to obtain accurate results. Therefore, this work proposes a workflow for relative quantitative PCR and introduces the relevant points so that beginners can better understand and use this technology.

Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time Sequencing.

Clinical-grade preparations of adeno-associated virus (AAV) vectors used for gene therapy typically undergo a series of diagnostics to determine titer, purity, homogeneity, and the presence of DNA contaminants. One type of contaminant that remains poorly investigated is replication-competent (rc)AAVs. rcAAVs form through recombination of DNA originating from production materials, yielding intact, replicative, and potentially infectious virus-like virions. They can be detected through the serial passaging of lysates from cells transduced by AAV vectors in the presence of wildtype adenovirus. Cellular lysates from the last passage are subjected to qPCR to detect the presence of the rep gene. Unfortunately, the method cannot be used to query the diversity of recombination events, nor can qPCR provide insights into how rcAAVs arise. Thus, the formation of rcAAVs through errant recombination events between ITR-flanked gene of interest (GOI) constructs and expression constructs carrying the rep-cap genes is poorly described. We have used single molecule, real-time sequencing (SMRT) to analyze virus-like genomes expanded from rcAAV-positive vector preparations. We present evidence that sequence-independent and non-homologous recombination between the ITR-bearing transgene and the rep/cap plasmid occurs under several events and rcAAVs spawn from diverse clones.