Wobble Editing of Cre-box by Unspecific CRISPR/Cas9 Causes CCR Release and Phenotypic Changes in Bacillus pumilus

CRISPR-associated Cas9 endonuclease (CRISPR/Cas9) systems are widely used to introduce precise mutations, such as knocking in/out at targeted genomic sites. Herein, we successfully disrupted the transcription of multiple genes in Bacillus pumilus LG3145 using a series of unspecific guide RNAs (gRNAs) and UgRNA:Cas9 system-assisted cre-box editing. The bases used as gRNAs shared 30–70% similarity with a consensus sequence, a cis-acting element (cre-box) mediating carbon catabolite repression (CCR) of many genes in Bacillus. This triggers trans-crRNA:Cas9 complex wobble cleavage up/downstream of cre sites in the promoters of multiple genes (up to 7), as confirmed by Sanger sequencing and next-generation sequencing (NGS). LG3145 displayed an obvious CCR release phenotype, including numerous secondary metabolites released into the culture broth, ∼ 1.67 g/L white flocculent protein, pigment overflow causing orange-coloured broth (absorbance = 309 nm), polysaccharide capsules appearing outside cells, improved sugar tolerance, and a two-fold increase in cell density. We assessed the relationship between carbon catabolite pathways and phenotype changes caused by unspecific UgRNA-directed cre site wobble editing. We propose a novel strategy for editing consensus targets at operator sequences that mediates transcriptional regulation in bacteria.

Download Full-text

Next-Generation Sequencing in Tumor Diagnosis and Treatment

Diagnostics ◽

10.3390/diagnostics10110962 ◽

2020 ◽

Vol 10 (11) ◽

pp. 962

Author(s):

Dario de Biase ◽

Matteo Fassan ◽

Umberto Malapelle

Keyword(s):

Next Generation Sequencing ◽

Tumor Diagnosis ◽

Diagnosis And Treatment ◽

Next Generation ◽

Multiple Genes ◽

Next Generation Sequencing Ngs ◽

Very High ◽

Generation Sequencing ◽

High Depth

Next-Generation Sequencing (NGS) allows for the sequencing of multiple genes at a very high depth of coverage [...]

Download Full-text

Abstract MP12: The Mef2c Transcription Factor Regulates The Renin Cell Identity

Hypertension ◽

10.1161/hyp.78.suppl_1.mp12 ◽

2021 ◽

Vol 78 (Suppl_1) ◽

Author(s):

Omar E Guessoum ◽

Kristyna Kupkova ◽

Nathan Sheffield ◽

Maria Luisa Sequeira Lopez ◽

Roberto A Gomez

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Consensus Sequence ◽

Renin Angiotensin System ◽

Fold Increase ◽

Conditional Knockout ◽

Motif Analysis ◽

Regulation Of Expression ◽

Cell Identity

Introduction: The Renin-Angiotensin-System is essential to maintain blood pressure and fluid electrolyte homeostasis. Because precise regulation of expression and release of renin is critical for survival, understanding the molecular regulation of the renin cell identity is a vital area of study. Advances in epigenetics have enabled finer dissection of chromatin factors which maintain the identity of the renin cell. By studying genes with heightened accessibility profiles that are unique to the JG cell, we now have the capacity to unravel the determinants of the renin cell identity. Hypothesis: That transcription factors central to the governance of renin cell identity can be identified through the Assay for Transposase Accessible Chromatin (ATAC-seq) differential accessibility analysis. Methods: Native renin cell ATAC-seq was compared to existing ENCODE ATAC-seq datasets from 40 other cell types to define regions/peaks which characterize the JG program. Peaks with high intensity and ≥2-fold increase in signal were selected for Motif analysis to search for transcription factors (TFs) whose consensus sequence is enriched in those regions. Identified TFs were then selected for validation by in-situ hybridization and conditional deletion in renin cells. Results: 1) The Mef2c transcription factor was identified as having a consensus sequence in regulatory regions unique to the JG cell. It has clear expression in RNA-seq of renin cells (65 transcripts per million, n=3) and a predicted binding site in the renin gene. These results were validated by in-situ hybridization where signal localized at the JG area was detected in concordance with our in-silico results. 2) We generated Mef2c conditional knockout animals using our Ren1d-Cre mouse to study the effect in renin expression and identity. These mice displayed reduced renin immunostaining at the JG area and a 40% reduction in renin mRNA expression by qPCR from kidney cortices relative to wild-type (n=2, preliminary data). Conclusions: Our studies identified Mef2c as a TF target which likely has an essential role in maintaining and preserving renin cell identity. Experiments involving transcriptomics and epigenomics are ongoing to understand the changes wrought by Mef2c deletion in renin cells.

Download Full-text

Vacuolar Sequestration of Azoles, a Novel Strategy of Azole Antifungal Resistance Conserved across Pathogenic and Nonpathogenic Yeast

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01347-18 ◽

2019 ◽

Vol 63 (3) ◽

Cited By ~ 9

Author(s):

Nitesh Kumar Khandelwal ◽

Mohd Wasi ◽

Remya Nair ◽

Meghna Gupta ◽

Mohit Kumar ◽

...

Keyword(s):

Abc Transporter ◽

Atp Hydrolysis ◽

Consensus Sequence ◽

Antifungal Resistance ◽

Azole Resistance ◽

Casein Kinase I ◽

Pathogenic Yeast ◽

Content Type ◽

Novel Strategy ◽

Vacuolar Sequestration

ABSTRACT Target alteration and overproduction and drug efflux through overexpression of multidrug transporters localized in the plasma membrane represent the conventional mechanisms of azole antifungal resistance. Here, we identify a novel conserved mechanism of azole resistance not only in the budding yeast Saccharomyces cerevisiae but also in the pathogenic yeast Candida albicans. We observed that the vacuolar-membrane-localized, multidrug resistance protein (MRP) subfamily, ATP-binding cassette (ABC) transporter of S. cerevisiae, Ybt1, could import azoles into vacuoles. Interestingly, the Ybt1 homologue in C. albicans, Mlt1p, could also fulfill this function. Evidence that the process is energy dependent comes from the finding that a Mlt1p mutant version made by converting a critical lysine residue in the Walker A motif of nucleotide-binding domain 1 (required for ATP hydrolysis) to alanine (K710A) was not able to transport azoles. Additionally, we have shown that, as for other eukaryotic MRP subfamily members, deletion of the conserved phenylalanine amino acid at position 765 (F765Δ) results in mislocalization of the Mlt1 protein; this mislocalized protein was devoid of the azole-resistant attribute. This finding suggests that the presence of this protein on vacuolar membranes is an important factor in azole resistance. Further, we report the importance of conserved residues, because conversion of two serines (positions 973 and 976, in the regulatory domain and in the casein kinase I [CKI] consensus sequence, respectively) to alanine severely affected the drug resistance. Hence, the present study reveals vacuolar sequestration of azoles by the ABC transporter Ybt1 and its homologue Mlt1 as an alternative strategy to circumvent drug toxicity among pathogenic and nonpathogenic yeasts.

Download Full-text

Alterations in the adenine-plus-thymine-rich region of CEN3 affect centromere function in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.68 ◽

1987 ◽

Vol 7 (1) ◽

pp. 68-75 ◽

Cited By ~ 40

Author(s):

A Gaudet ◽

M Fitzgerald-Hayes

Keyword(s):

Saccharomyces Cerevisiae ◽

Base Composition ◽

Consensus Sequence ◽

Fold Increase ◽

Base Pairs ◽

Yeast Saccharomyces Cerevisiae ◽

Core Element ◽

Sequence Elements ◽

Dna Elements ◽

Chromosome Iii

Centromere DNA from 11 of the 16 chromosomes of the yeast Saccharomyces cerevisiae have been analyzed and reveal three sequence elements common to each centromere, referred to as conserved centromere DNA elements (CDE). The adenine-plus-thymine (A + T)-rich central core element, CDE II, is flanked by two short conserved sequences, CDE I (8 base pairs [bp]) and CDE III (25 bp). Although no consensus sequence exists among the different CDE II regions, they do have three common features of sequence organization. First, the CDE II regions are similar in length, ranging from 78 to 86 bp measured from CDE I to the left boundary of CDE III. Second, the base composition is always greater than 90% A + T. Finally, the A and T residues in these segments are often arranged in runs of A and runs of T residues, sometimes with six or seven bases in a stretch. We constructed insertion, deletion, and replacement mutations in the CDE II region of the centromere from chromosome III, CEN3, designed to investigate the length and sequence requirements for function of the CDE II region of the centromere. We analyzed the effect of these altered centromeres on plasmid and chromosome segregation in S. cerevisiae. Our results show that increasing the length of CDE II from 84 to 154 bp causes a 100-fold increase in chromosome nondisjunction. Deletion mutations removing segments of the A + T-rich CDE II DNA also cause aberrant segregation. In some cases partial function could be restored by replacing the deleted DNA with fragments whose primary sequence or base composition is very different from that of the wild-type CDE II DNA. In addition, we found that identical mutations introduced into different positions in CDE II have very similar effects.

Download Full-text

Human Argonaute2 and Argonaute3 are catalytically activated by different lengths of guide RNA

10.1101/2020.07.16.207720 ◽

2020 ◽

Author(s):

Mi Seul Park ◽

GeunYoung Sim ◽

Audrey C. Kehling ◽

Kotaro Nakanishi

Keyword(s):

Gene Silencing ◽

Fold Increase ◽

Specific Gene ◽

Small Interfering Rnas ◽

Guide Rna ◽

Guide Rnas ◽

Argonaute Proteins ◽

Complementary Rnas ◽

Standing Question

AbstractRNA interfering is a eukaryote-specific gene silencing by 20∼23 nucleotide (nt) microRNAs and small interfering RNAs that recruit Argonaute proteins to complementary RNAs for degradation. In humans, Argonaute2 (AGO2) has been known as the only slicer while Argonaute3 (AGO3) barely cleaves RNAs. Therefore, the intrinsic slicing activity of AGO3 remains controversial and a long-standing question. Here, we report 14-nt 3′ end-shortened variants of let-7a, miR-27a, and specific miR-17-92 families that make AGO3 an extremely competent slicer by an ∼ 82-fold increase in target cleavage. These RNAs, named cleavage-inducing tiny guide RNAs (cityRNAs), conversely lower the activity of AGO2, demonstrating that AGO2 and AGO3 have different optimum guide lengths for target cleavage. Our study sheds light on the role of tiny guide RNAs.

Download Full-text

Crispra: A Promising Tool for β-Thalassemia Treatment

Blood ◽

10.1182/blood-2020-141196 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 8-8

Author(s):

Xiaoqing Jia ◽

Qi Yao ◽

Hui Li ◽

Jieping Chen

Keyword(s):

Conflicts Of Interest ◽

Gene Activation ◽

Fold Increase ◽

293T Cell ◽

Genomic Locus ◽

Hematopoietic Stem ◽

Nb4 Cells ◽

Guide Rnas ◽

Promising Tool

Induction of hemoglobin γ expression is a reliable strategy to treat β-thalassemia. Gene editing using CRISPR/Cas9 technology has been widely used. However, application in vivo is limited due to the uncertainty on genomic cleavages of Cas9. In contrast, CRISPR/Cas9-based gene activation (CRISPRa) can only locate genomic locus but not interrupt sequence. Here, we use SAM system of CRISPRa to locate and activate HBG1 and HBG2, exploring the great potential of CRISPRa for β-thalassemia treatment. WWe designed 8 single-guide RNAs (sgRNAs) online and cloned into vector SAM V2, which fused dCas9 and VP64. To test the over-expression efficiency, vector containing sgRNA and MPH (fused HSF1, p65 and MS2) were transfected into 293T cell. After 72h transfection, 293T cells were collected. Q-PCR data showed that two sgRNAs were excellent on activating HBG expression with over 1000-fold increase. WTo test the activating function in hematological cell and the persistence of hemoglobin γexpression, two screened sgRNA were transfected into NB4 cells using lentivirus system. We harvested NB4 cells at different time-point (3 day, 1 week and 2 weeks), and implemented q-PCR assay. HBG expression were increased 50-hold and 1000-hold, respectively. However, the expression were reducing over time and the intrinsic mechanism is unknown. WThis study set out to increase HBG without interrupt genome using CRISPRa system. This study has found two sgRNA to activate the expression of HBG in 293T cell and NB4 cell. Further research is required to vertify the efficiency of sgRNA in hematopoietic stem cells and prolong the expression time. Figure Disclosures No relevant conflicts of interest to declare.

Download Full-text

Early Clonal Hematopoiesis Detected in Gata2 heterozygosity By Color-Barcoding of Hematopoietic Stem Cells in Zebrafish

Blood ◽

10.1182/blood-2018-99-118236 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 748-748

Author(s):

Serine Avagyan ◽

William P. Mannherz ◽

Leonard I. Zon

Keyword(s):

Conflicts Of Interest ◽

Myeloid Cell ◽

Fold Increase ◽

Shannon Index ◽

Hematopoietic Stem ◽

Guide Rnas ◽

Clonal Hematopoiesis ◽

Children And Young Adults ◽

Epigenetic Regulators ◽

Different Color

Abstract Germline GATA2 haploinsufficiency underlies an inherited syndrome of predisposition to myeloid malignancies. The mechanism by which GATA2 mutations lead to leukemia in children and young adults remains to be determined. We generated gata2b heterozygous mutant zebrafish using CRISPR/Cas9 technology to study this process. We used a color-barcoding system in zebrafish called Zebrabow that labels each hematopoietic stem cell (HSC) born during embryogenesis with a different color. Color-barcoding was induced by a blood-specific promoter-driven Cre recombinase during development at 24 hours post fertilization when there are on average 20 HSC clones. We then examined adult hematopoiesis in wildtype and gata2b+/- zebrafish. Surprisingly, at steady state the marrow of gata2b+/- fish showed color dominance with a single color clone contributing to over 30% of granulocytes, while the myeloid output in wildtype clutchmates was polyclonal. This suggested a baseline oligoclonal state in gata2b+/- hematopoiesis. Gata2b+/- fish also had a concomitant mild myelocytopenia with 20% reduction of myeloid cells in the marrow as early as 2 months post fertilization (mpf), reminescent of monocytopenia often present in GATA2 patients. Myelocytopenia with concurrent expansion of progenitor population was more profound in gata2b-/- fish with up to 4-fold increase of progenitor to myeloid cell ratio at 3mpf. Gata2b-/- fish are adult viable, possibly due to partial compensation by a duplicated gata2a gene. Germline GATA2-associated MDS and AML often harbor additional acquired mutations in epigenetic regulators. To study the effect of somatic mutations in GATA2 heterozygosity, we induced mosaic mutagenesis in wild-type or gata2b+/-embryos by injecting 1-cell embryos with Cas9 mRNA and guide RNAs targeting zebrafish orthologs of ASXL1 and STAG2, both of which have been associated with GATA2 mutations in MDS or AML. The marrow analysis of injected gata2b+/- fish showed an increase of hematopoietic progenitor cells and worse myelocytopenia at 7 mpf compared to control injected wildtype fish (progenitor/myeloid ratio of 1.7, p < 0.05). Single color expansion was observed in over 60% of injected gata2b+/-fish as measured by Shannon index of diversity, and correlated with stag2a and stag2b frame-shift mutations with variant allele fraction of >20%. This mutations result in the predicted loss of at least 2 of the 4 total stag2 alleles. In summary, gata2b deficiency alone results in aberrant hematopoiesis with reduced HSC diversity and myelocytopenia. Mutations in stag2 lead to early enhanced clonal dominance in gata2b+/- but not wildtype fish. Our studies suggest a model in which germline GATA2 mutations lead to a state of reduced HSC diversity during development. Such a state predisposes to clonal events due to acquired mutations in epigenetic regulators resulting in clonal hematopoiesis and myeloid leukemias, establishing a link between abnormal developmental hematopoiesis and leukemia risk later in life. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Fibroblast transition to an endothelial “trans” state improves cell reprogramming efficiency

Scientific Reports ◽

10.1038/s41598-021-02056-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Megumi Mathison ◽

Deepthi Sanagasetti ◽

Vivek P. Singh ◽

Aarthi Pugazenthi ◽

Jaya Pratap Pinnamaneni ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cardiac Fibroblasts ◽

Fold Increase ◽

Differentiation Potential ◽

Neonatal Cardiomyocytes ◽

Cell Transdifferentiation ◽

Reprogramming Efficiency ◽

Novel Strategy ◽

Human Cardiac Fibroblasts

AbstractFibroblast reprogramming offers the potential for myocardial regeneration via in situ cell transdifferentiation. We explored a novel strategy leveraging endothelial cell plasticity to enhance reprogramming efficiency. Rat cardiac endothelial cells and fibroblasts were treated with Gata4, Mef2c, and Tbx5 (GMT) to assess the cardio-differentiation potential of these cells. The endothelial cell transdifferentiation factor ETV2 was transiently over-expressed in fibroblasts followed by GMT treatment to assess “trans-endothelial” cardio-differentiation. Endothelial cells treated with GMT generated more cTnT+ cells than did cardiac fibroblasts (13% ± 2% vs 4% ± 0.5%, p < 0.01). Cardiac fibroblasts treated with ETV2 demonstrated increased endothelial cell markers, and when then treated with GMT yielded greater prevalence of cells expressing cardiomyocyte markers including cTnT than did fibroblasts treated with GMT or ETV2 (10.3% ± 0.2% vs 1.7% ± 0.06% and 0.6 ± 0.03, p < 0.01). Rat cardiac fibroblasts treated with GMT + ETV2 demonstrated calcium transients upon electrical stimulation and contractility synchronous with surrounding neonatal cardiomyocytes, whereas cells treated with GMT or ETV2 alone failed to contract in co-culture experiments. Human cardiac fibroblasts treated with ETV2 and then GMT likewise demonstrated greater prevalence of cTnT expression than did cells treated with GMT alone (2.8-fold increase, p < 0.05). Cardiac fibroblast transitioning through a trans-endothelial state appears to enhance cardio-differentiation by enhancing fibroblast plasticity.

Download Full-text

Do degree and rate of silicate weathering depend on plant productivity?

Biogeosciences ◽

10.5194/bg-17-4883-2020 ◽

2020 ◽

Vol 17 (19) ◽

pp. 4883-4917 ◽

Cited By ~ 1

Author(s):

Ralf A. Oeser ◽

Friedhelm von Blanckenburg

Keyword(s):

Primary Productivity ◽

Net Primary Productivity ◽

Nutrient Recycling ◽

Fold Increase ◽

Critical Zone ◽

Mineral Nutrient ◽

Silicate Weathering ◽

Weathering Rates ◽

Novel Strategy ◽

The Impact

Abstract. Plants and their associated below-ground microbiota possess the tools for rock weathering. Yet the quantitative evaluation of the impact of these biogenic weathering drivers relative to abiogenic parameters, such as the supply of primary minerals, water, and acids, is an open question in Critical Zone research. Here we present a novel strategy to decipher the relative impact of these drivers. We quantified the degree and rate of weathering and compared these to nutrient uptake along the “EarthShape” transect in the Chilean Coastal Cordillera. These sites define a major north–south gradient in precipitation and primary productivity but overlie granitoid rock throughout. We present a dataset of the chemistry of Critical Zone compartments (bedrock, regolith, soil, and vegetation) to quantify the relative loss of soluble elements (the “degree of weathering”) and the inventory of bioavailable elements. We use 87Sr∕86Sr isotope ratios to identify the sources of mineral nutrients to plants. With rates from cosmogenic nuclides and biomass growth we determined fluxes (“weathering rates”), meaning the rate of loss of elements out of the ecosystems, averaged over weathering timescales (millennia), and quantified mineral nutrient recycling between the bulk weathering zone and the bulk vegetation cover. We found that neither the degree of weathering nor the weathering rates increase systematically with precipitation from north to south along the climate and vegetation gradient. Instead, the increase in biomass nutrient demand is accommodated by faster nutrient recycling. In the absence of an increase in weathering rate despite a five-fold increase in precipitation and net primary productivity (NPP), we hypothesize that plant growth might in fact dampen weathering rates. Because plants are thought to be key players in the global silicate weathering–carbon feedback, this hypothesis merits further evaluation.

Download Full-text

Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the FAD2 and FATB Multigene in Soybean

Plants ◽

10.3390/plants10112542 ◽

2021 ◽

Vol 10 (11) ◽

pp. 2542

Author(s):

Won-Nyeong Kim ◽

Hye-Jeong Kim ◽

Young-Soo Chung ◽

Hyun-Uk Kim

Keyword(s):

Reverse Genetics ◽

Restriction Enzymes ◽

Golden Gate ◽

Knock Out ◽

Guide Rnas ◽

Genetics Research ◽

Multiple Genes ◽

Cas9 Protein ◽

Cloning Method ◽

Golden Gate Cloning

CRISPR/Cas9 is a commonly used technique in reverse-genetics research to knock out a gene of interest. However, when targeting a multigene family or multiple genes, it is necessary to construct a vector with multiple single guide RNAs (sgRNAs) that can navigate the Cas9 protein to the target site. In this protocol, the Golden Gate cloning method was used to generate multiple sgRNAs in the Cas9 vector. The vectors used were pHEE401E_UBQ_Bar and pBAtC_tRNA, which employ a one-promoter/one-sgRNA and a polycistronic-tRNA-gRNA strategy, respectively. Golden Gate cloning was performed with type IIS restriction enzymes to generate gRNA polymers for vector inserts. Four sgRNAs containing the pHEE401E_UBQ_Bar vector and four to six sgRNAs containing the pBAtC_tRNA vector were constructed. In practice, we constructed multiple sgRNAs targeting multiple genes of FAD2 and FATB in soybean using this protocol. These three vectors were transformed into soybeans using the Agrobacterium-mediated method. Using deep sequencing, we confirmed that the T0 generation transgenic soybean was edited at various indel ratios in the predicted target regions of the FAD2 and FATB multigenes. This protocol is a specific guide that allows researchers to easily follow the cloning of multiple sgRNAs into commonly used CRISPR/Cas9 vectors for plants.

Download Full-text