Display omitted
•Chaperone and CRISPRi were first applied in algae under thermophilic condition.•Transcriptional result depicts temperature and CRISPRi effects on algal metabolism.•PGi strain showed ...2.56 g/L, 23.5 and 893 mg/L of biomass, lutein and lipid at 35°C.•Thermophilic conditions affect the biomass, lipid, and CO2 uptake in microalgae.•PGi strain had 1.08 g-CO2/g-DCW of CO2 assimilation ability in mixotrophic culture.
Microalgae are widely recognized as a promising bioresource for producing renewable fuels and chemicals. Microalgal biorefinery has tremendous potential for incorporation into circular bioeconomy, including sustainability, cascading use, and waste reduction. In this study, genetic engineering was used to enhance the growth, lipid and lutein productivity of Chlamydomonas reinhardtii including strains of CC400, PY9, pCHS, and PG. Notably, CRISPRi mediated on phosphoenolpyruvate carboxylase (PEPC1) gene to down-regulate the branch pathway from glycolysis to partitioning more carbon flux to lipid was explored under meso-thermophilic condition. The best chassis PGi, which has overexpressed chaperone GroELS and applied CRISPRi resulting in the highest biomass of 2.56 g/L and also boosted the lipids and lutein with 893 and 23.5 mg/L, respectively at 35°C. Finally, all strains with CRISPRi exhibited higher transcriptional levels of the crucial genes from photosynthesis, starch, lipid and lutein metabolism, thus reaching a CO2 assimilation of 1.087 g-CO2/g-DCW in mixotrophic condition.
BackgroundThe pathological HTT CAG repeat continues to expand throughout life. As CAG repeat length predicts rate of expansion and age of HD onset, slowing expansion is an attractive therapeutic ...approach. The GeM-HD GWASs associated changes in age of onset with many loci, including DNA damage repair genes. Understanding if/how these risk modifiers affect repeat expansion rate, and their interplay in vulnerable neurons, will contribute to understanding the underlying expansion mechanism.Aims1) Generate a human HD iPSC-based model capable of rapidly assaying risk associated genes for effects on somatic expansion of the HTT CAG repeat in striatal neuron cultures. 2) Target expression of components of the mismatch repair MutL complex (MLH1, PMS1, MLH3 & PMS2) as validation.MethodsdCas9 fused to a transcriptional repressor was knocked-in to the CLYBL safe harbour locus of the 125Q iPSC juvenile HD line. Guides targeting individual MutL components were introduced by lentiviral transduction. Knock-down pools for each gene were differentiated to post-mitotic MSNs and assayed for CAG expansion.ResultsKnock-in clones retained parental CAG expansion rates and differentiation potential. CRISPRi achieved 70-90% reduction in target expression. Knock-down of the individual MutL proteins suppressed expansion in striatal neuron cultures.ConclusionsThis model successfully recapitulates previously reported effects of MutL components on somatic expansion of the HTT CAG repeat. This indicates our model will be a useful platform for rapidly dissecting the effects of other risk modifier gene expression on CAG instability and pathophysiology in a human model of HD.
The methylotrophic yeast Komagataella phaffii is a popular host system for the pharmaceutical and biotechnological production of recombinant proteins. CRISPR-Cas9 and its derivative CRISPR ...interference (CRISPRi) offer a promising avenue to further enhance and exploit the full capabilities of this host. MAD7 and its catalytically inactive variant “dead” MAD7 (dMAD7) represent an interesting alternative to established CRISPR-Cas9 systems and are free to use for industrial and academic research. CRISPRi utilizing dMAD7 does not introduce double-strand breaks but only binds to the DNA to regulate gene expression. Here, we report the first use of dMAD7 in K. phaffii to regulate the expression of the enhanced green fluorescent protein (eGFP). A reduction of eGFP fluorescence level (up to 88%) was achieved in random integration experiments using dMAD7 plasmids. Integration loci/events of investigated strains were assessed through whole genome sequencing. Additionally, RNA-sequencing experiments corroborated the whole genome sequencing results and showed a significantly reduced expression of eGFP in strains containing a dMAD7 plasmid, among others. Our findings conclusively demonstrate the utility of dMAD7 in K. phaffii through successfully regulating eGFP expression.
•We report the first use of dMAD7 in K. phaffii to regulate the expression of enhanced green fluorescent protein (eGFP).•A reduction of eGFP fluorescence level (up to 88%) was achieved in random integration experiments using dMAD7 plasmids.•Whole genome sequencing and RNA-sequencing were used to assess the integration events and to characterize the transformed strains.•Our findings conclusively demonstrate the utility of the dMAD7 system in K. phaffii.
Zymomonas mobilis is a promising biofuel producer due to its high alcohol tolerance and streamlined metabolism that efficiently converts sugar to ethanol. Z. mobilis genes are poorly characterized ...relative to those of model bacteria, hampering our ability to rationally engineer the genome with pathways capable of converting sugars from plant hydrolysates into valuable biofuels and bioproducts. Many of the unique properties that make Z. mobilis an attractive biofuel producer are controlled by essential genes; however, these genes cannot be manipulated using traditional genetic approaches (e.g., deletion or transposon insertion) because they are required for viability. CRISPR interference (CRISPRi) is a programmable gene knockdown system that can precisely control the timing and extent of gene repression, thus enabling targeting of essential genes. Here, we establish a stable, high-efficacy CRISPRi system in Z. mobilis that is capable of perturbing all genes—including essential genes. We show that Z. mobilis CRISPRi causes either strong knockdowns (>100-fold) using single guide RNA (sgRNA) spacers that perfectly match target genes or partial knockdowns using spacers with mismatches. We demonstrate the efficacy of Z. mobilis CRISPRi by targeting essential genes that are universally conserved in bacteria, are key to the efficient metabolism of Z. mobilis, or underlie alcohol tolerance. Our Z. mobilis CRISPRi system will enable comprehensive gene function discovery, opening a path to rational design of biofuel production strains with improved yields.
Bacteriophage φX174 is a small icosahedral virus of the Microviridae with a rapid replication cycle. Previously, we found that in φX174 infections of Escherichia coli, the most highly upregulated ...host proteins are two small heat shock proteins, IbpA and IbpB, belonging to the HSP20 family, which is a universally conserved group of stress-induced molecular chaperones that prevent irreversible aggregation of proteins. Heat shock proteins were found to protect against φX174 lysis, but IbpA/B have not been studied. In this work, we disrupted the ibpA and ibpB genes and measured the effects on φX174 replication. We found that in contrast to other E. coli heat shock proteins, they are not necessary for φX174 replication; moreover, their absence has no discernible effect on φX174 fecundity. These results suggest IbpA/B upregulation is a response to φX174 protein expression but does not play a role in phage replication, and they are not Microviridae host factors.
•Small heat shock protein genes ibpAB are collectively non-essential for phiX174 replication.•Individual ibpA or ibpB gene knockouts in E. coli C result in heat sensitivity at 45°C.•In contrast to K-12 strain backgrounds, the ibpAB genes may be collectively essential for E. coli C strains.
Over one million candidate regulatory elements have been identified across the human genome, but nearly all are unvalidated and their target genes uncertain. Approaches based on human genetics are ...limited in scope to common variants and in resolution by linkage disequilibrium. We present a multiplex, expression quantitative trait locus (eQTL)-inspired framework for mapping enhancer-gene pairs by introducing random combinations of CRISPR/Cas9-mediated perturbations to each of many cells, followed by single-cell RNA sequencing (RNA-seq). Across two experiments, we used dCas9-KRAB to perturb 5,920 candidate enhancers with no strong a priori hypothesis as to their target gene(s), measuring effects by profiling 254,974 single-cell transcriptomes. We identified 664 (470 high-confidence) cis enhancer-gene pairs, which were enriched for specific transcription factors, non-housekeeping status, and genomic and 3D conformational proximity to their target genes. This framework will facilitate the large-scale mapping of enhancer-gene regulatory interactions, a critical yet largely uncharted component of the cis-regulatory landscape of the human genome.
Display omitted
•Perturbed 5,920 human candidate enhancers for impact on gene expression•Multiplexed ∼28 CRISPRi perturbations per single-cell transcriptome•Adapted the eQTL analytical framework to identify 664 cis human enhancer-gene pairs•Characterized genomic features associated with these enhancer-gene pairs
A highly multiplexed CRISPRi screen uncovers gene-enhancer relationships at scale.
Programmable control of gene expression is essential to understanding gene function, engineering cellular behaviors, and developing therapeutics. Beyond the gene editing applications enabled by the ...nuclease CRISPR–Cas9 and CRISPR–Cas12a, the invention of the nuclease-dead Cas molecules (dCas9 and dCas12a) offers a platform for the precise control of genome function without gene editing. Diverse dCas tools have been developed, which constitute a comprehensive toolbox that allows for interrogation of gene function and modulation of the cellular behaviors. This review summarizes current applications of the dCas tools for transcription regulation, epigenetic engineering, genome imaging, genetic screens, and chromatin immunoprecipitation. We also highlight the advantages and existing challenges of the current dCas tools in genetic engineering and synthetic biology, and provide perspectives on future directions and applications.
Display omitted
•The dCas system is a programmable and versatile platform for gene regulation and epigenome control.•Focus on advances of tool development and synthetic biology applications of the dCas platform•Highlight using dCas for transcription, epigenetics, imaging, screening, and ChIP•Summarize advantages, challenges, and perspectives of the dCas system as a technology platform
5-Methyltetrahydrofolate (5-MTHF) is the predominant folate form in human plasma, which has been widely used as a nutraceutical. However, the microbial synthesis of 5-MTHF is currently inefficient, ...limiting green and sustainable 5-MTHF production. In this study, the Generally Regarded As Safe (GRAS) microorganism Bacillus subtilis was engineered as the 5-MTHF production host. Three precursor supply modules were first optimized by modular engineering for strengthening the supply of guanosine-5-triphosphate (GTP) and p-aminobenzoic acid (pABA). Next, the impact of genome-wide gene expression on 5-MTHF biosynthesis was evaluated using transcriptome analyses, which identified key genes for 5-MTHF production. The effects of potential genes on 5-MTHF synthesis were verified by observing the genes’ up-regulated by strong promoter P566 and those down-regulated by inhibition through the clustered regularly interspaced short palindromic repeat interference (CRISPRi). Finally, a key gene for improved 5-MTHF biosynthesis, comGC, was integrated into the genome of modular engineered strain B89 for its overexpression and facilitating efficient 5-MTHF synthesis, reaching 3.41 ± 0.10 mg/L with a productivity of 0.21 mg/L/h, which was the highest level achieved by microbial synthesis. The engineered 5-MTHF-producing B. subtilis developed in this work lays the foundation of further enhancing 5-MTHF production by microbial fermentation, which can be used for isolation and purification of 5-MTHF as food and nutraceutical ingredients.
Noncoding mutations in cancer genomes are frequent but challenging to interpret. PVT1 encodes an oncogenic lncRNA, but recurrent translocations and deletions in human cancers suggest alternative ...mechanisms. Here, we show that the PVT1 promoter has a tumor-suppressor function that is independent of PVT1 lncRNA. CRISPR interference of PVT1 promoter enhances breast cancer cell competition and growth in vivo. The promoters of the PVT1 and the MYC oncogenes, located 55 kb apart on chromosome 8q24, compete for engagement with four intragenic enhancers in the PVT1 locus, thereby allowing the PVT1 promoter to regulate pause release of MYC transcription. PVT1 undergoes developmentally regulated monoallelic expression, and the PVT1 promoter inhibits MYC expression only from the same chromosome via promoter competition. Cancer genome sequencing identifies recurrent mutations encompassing the human PVT1 promoter, and genome editing verified that PVT1 promoter mutation promotes cancer cell growth. These results highlight regulatory sequences of lncRNA genes as potential disease-associated DNA elements.
Display omitted
•Silencing PVT1 promoter enhances breast cancer cell competition•PVT1 promoter inhibits MYC transcription independent of PVT1 lncRNA•PVT1 and MYC promoters compete for enhancer contact in cis•Mutations encompassing PVT1 promoter are recurrent in human cancers
Recurrent mutations in human cancer are found encompassing the promotor for the lncRNA gene PVT1, which regulates MYC transcription via promoter competition for a shared set of enhancers.
PDF
