scholarly journals Effective CRISPRa-Mediated Control of Gene Expression in Bacteria Must Overcome Strict Target Site Requirements

2019 ◽  
Author(s):  
Jason Fontana ◽  
Chen Dong ◽  
Cholpisit Kiattisewee ◽  
Venkata P. Chavali ◽  
Benjamin I. Tickman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Target Site ◽  
Multiple Features ◽  
Control Of Gene Expression ◽  
Regulate Gene Expression ◽  
Guide Rna ◽  
Target Sites ◽  
Helical Turn ◽  
The Right

AbstractIn bacterial systems, CRISPR-Cas transcriptional activation (CRISPRa) has the potential to dramatically expand our ability to regulate gene expression, but we currently lack a complete understanding of the rules for designing effective guide RNA target sites. We have identified multiple features of bacterial promoters that impose stringent requirements on bacterial CRISPRa target sites. Most importantly, we found that shifting a gRNA target site by 2-4 bases along the DNA target can cause a nearly complete loss in activity. The loss in activity can be rescued by shifting the target site 10-11 bases, corresponding to one full helical turn. Practically, our results suggest that it will be challenging to find a gRNA target site with an appropriate PAM sequence at precisely the right position at arbitrary genes of interest. To overcome this limitation, we demonstrate that a dCas9 variant with expanded PAM specificity allows activation of promoters that cannot be activated by S. pyogenes dCas9. These results provide a roadmap for future engineering efforts to further expand and generalize the scope of bacterial CRISPRa.

scholarly journals A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana.

2021 ◽  
Author(s):  
Elena Garcia-Perez ◽  
Borja Diego-Martin ◽  
Alfredo Quijano-Rubio ◽  
Elena Moreno Gimenez ◽  
Diego Orzaez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Nicotiana Benthamiana ◽  
Gene Networks ◽  
Rna Binding ◽  
Gene Activation ◽  
Dependent Manner ◽  
Copper Binding ◽  
Control Of Gene Expression ◽  
Guide Rna

CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS). In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations in place, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold for the endogenous N. benthamiana DFR gene, with negligible expression in the absence of the trigger. The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.

scholarly journals Waves of chromatin modifications in mouse dendritic cells in response to LPS stimulation

2016 ◽  
Author(s):  
Alexis Vandenbon ◽  
Yutaro Kumagai ◽  
Yutaka Suzuki ◽  
Kenta Nakai
Keyword(s):  
Gene Expression ◽  
Dendritic Cells ◽  
Histone Modifications ◽  
Transcriptional Activation ◽  
Control Of Gene Expression ◽  
Time Series Dataset ◽  
Binding Data ◽  
A Genome ◽  
Extracellular Stimuli ◽  
Wide Scale

AbstractBackgroundThe importance of transcription factors (TFs) and epigenetic modifications in the control of gene expression is widely accepted. However, causal relationships between changes in TF binding, histone modifications, and gene expression during the response to extracellular stimuli are not well understood. Here, we analyzed the ordering of these events on a genome-wide scale in dendritic cells (DCs) in response to lipopolysaccharide (LPS) stimulation.ResultsUsing a ChIP-seq time series dataset, we found that the LPS-induced accumulation of different histone modifications follow clearly distinct patterns. Increases in H3K4me3 appear to coincide with transcriptional activation. In contrast, H3K9K14ac accumulates early after stimulation, and H3K36me3 at later time points. Integrative analysis with TF binding data revealed potential links between TF activation and dynamics in histone modifications. Especially, LPS-induced increases in H3K9K14ac and H3K4me3 were associated with binding by STAT1/2, and were severely impaired inStat1-/-cells.ConclusionsWhile the timing of short-term changes of some histone modifications coincides with changes in transcriptional activity, this is not the case for others. In the latter case, dynamics in modifications more likely reflect strict regulation by stimulus-induced TFs, and their interactions with chromatin modifiers.

Engineered protein switches for exogenous control of gene expression

2020 ◽  
Vol 48 (5) ◽  
pp. 2205-2212
Author(s):  
Shaun Spisak ◽  
Marc Ostermeier
Keyword(s):  
Gene Expression ◽  
Domain Swapping ◽  
Signaling Proteins ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Regulate Gene Expression ◽  
Substrate Specificities ◽  
Large Pool ◽  
Domain Insertion ◽  
Exogenous Control

There is an ongoing need in the synthetic biology community for novel ways to regulate gene expression. Protein switches, which sense biological inputs and respond with functional outputs, represent one way to meet this need. Despite the fact that there is already a large pool of transcription factors and signaling proteins available, the pool of existing switches lacks the substrate specificities and activities required for certain applications. Therefore, a large number of techniques have been applied to engineer switches with novel properties. Here we discuss some of these techniques by broadly organizing them into three approaches. We show how novel switches can be created through mutagenesis, domain swapping, or domain insertion. We then briefly discuss their use as biosensors and in complex genetic circuits.

scholarly journals Dimerization is required for transactivation by estrogen-receptor-related (ERR) orphan receptors: evidence from amphioxus ERR

2004 ◽  
Vol 33 (2) ◽  
pp. 493-509 ◽  
Author(s):  
B Horard ◽  
A Castet ◽  
P-L Bardet ◽  
V Laudet ◽  
V Cavailles ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Nuclear Receptor ◽  
Transcriptional Activation ◽  
Target Site ◽  
Estrogen Response Element ◽  
Orphan Receptors ◽  
Response Element ◽  
Nuclear Receptor Superfamily ◽  
Estrogen Response ◽  
Target Sites

The estrogen-receptor-related (ERR) receptors are orphan members of the nuclear receptor superfamily that bind to their specific DNA target sites as homodimers. However, it has not been shown whether this mode of binding is required for the transcriptional activation they drive. We here show that heterodimerization can also occur between these receptors. Furthermore, we demonstrate that the unique amphioxus ortholog of ERR genes (AmphiERR) is expressed as two isoforms differing by an in-frame insertion. While the short isoform behaves like its mammalian counterparts, the long isoform (AmphiERR(L)) displays divergent transcriptional properties according to the target site to which it binds. Indeed, AmphiERR(L) binds as a monomer but does not activate transcription through the SF1 response element (SFRE). On the contrary, this isoform binds as a homodimer and activates transcription through the classical estrogen-response element. Our results strongly suggest that dimerization is required for transactivation exerted by the ERR receptors.

scholarly journals CRISPR/Cas “non-target” sites inhibit on-target cutting rates

2020 ◽  
Author(s):  
Eirik A. Moreb ◽  
Mitchell Hutmacher ◽  
Michael D. Lynch
Keyword(s):  
Genome Editing ◽  
Target Site ◽  
High Fidelity ◽  
List Type ◽  
Dependent Manner ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Target Sites ◽  
Dose Dependent ◽  
Target Activity

AbstractCRISPR/Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 associated with a guide RNA (gRNA) searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM) and once found, cuts the DNA. The number of PAM sites in the genome are effectively a non-target pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity towards a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates. Decreasing the non-target pool by increasing PAM specificity provides a path towards improving on-target activity for slower high fidelity Cas9 variants. These results demonstrate the importance of competitive non-target sites on Cas9 activity and, in part, may help to explain sequence and context dependent activities of gRNAs. Engineering improved PAM specificity to reduce the competitive non-target pool offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.HighlightsThe pool of non-target PAM sites inhibit Cas9/gRNA on-target activitynon-target PAM inhibition is dose dependentnon-target PAM inhibition is a function of gRNA sequencenon-target PAM inhibition is a function of Cas9 levels

scholarly journals Engineered CRISPR-Cas9 system enables noiseless, fine-tuned and multiplexed repression of bacterial genes

2017 ◽  
Author(s):  
Antoine Vigouroux ◽  
Enno Oldewurtel ◽  
Lun Cui ◽  
Sven van Teeffelen ◽  
David Bikard
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Genetic Regulation ◽  
Copy Number Variations ◽  
Control Of Gene Expression ◽  
Guide Rna ◽  
Cas9 Nuclease ◽  
Feedback Strength ◽  
Bacterial Genes ◽  
The Impact

AbstractOver the past few years, tools that make use of the Cas9 nuclease have led to many breakthroughs, including in the control of gene expression. The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to block transcription of target genes, in a strategy also known as CRISPRi. Here, we reveal that the level of complementarity between the guide RNA and the target controls the rate at which dCas9 successfully blocks the RNA polymerase. We use this mechanism to precisely and robustly reduce gene expression by defined relative amounts. We demonstrate broad applicability of this method to the study of genetic regulation and cellular physiology. First, we characterize feedback strength of a model auto-repressor. Second, we study the impact of copy-number variations of cell-wall synthesizing enzymes on cell morphology. Finally, we demonstrate that this system can be multiplexed to obtain any combination of fractional repression of two genes.

scholarly journals Amino Acids Control Mammalian Gene Transcription: Activating Transcription Factor 2 Is Essential for the Amino Acid Responsiveness of the CHOP Promoter

2000 ◽  
Vol 20 (19) ◽  
pp. 7192-7204 ◽  
Author(s):  
Alain Bruhat ◽  
Céline Jousse ◽  
Valérie Carraro ◽  
Andreas M. Reimold ◽  
Marc Ferrara ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Transcription Factor ◽  
Amino Acid ◽  
Transcriptional Activation ◽  
Dominant Negative Mutant ◽  
Transient Expression Assay ◽  
Control Of Gene Expression ◽  
Mammalian Gene ◽  
Activating Transcription Factor

ABSTRACT In mammals, plasma concentration of amino acids is affected by nutritional or pathological conditions. It has been well established that nutrients, and particularly amino acids, are involved in the control of gene expression. Here we examined the molecular mechanisms involved in the regulation ofCHOP (a CCAAT/enhancer-binding protein [C/EBP]-related gene) expression upon amino acid limitation. We have previously shown that regulation of CHOP mRNA expression by amino acid concentration has both transcriptional and posttranscriptional components. We report the analysis ofcis- and trans-acting elements involved in the transcriptional activation of the human CHOPgene by leucine starvation. Using a transient expression assay, we show that a cis-positive element is essential for amino acid regulation of the CHOP promoter. This sequence is the first described that can regulate a basal promoter in response to starvation for several individual amino acids and therefore can be called an amino acid response element (AARE). In addition, we show that the CHOP AARE is related to C/EBP and ATF/CRE binding sites and binds in vitro the activating transcription factor 2 (ATF-2) in starved and unstarved conditions. Using ATF-2-deficient mouse embryonic fibroblasts and an ATF-2-dominant negative mutant, we demonstrate that expression of this transcription factor is essential for the transcriptional activation of CHOP by leucine starvation. Altogether, these results suggest that ATF-2 may be a member of a cascade of molecular events by which the cellular concentration of amino acids can regulate mammalian gene expression.

scholarly journals The IS1111 Family Members IS4321 and IS5075 Have Subterminal Inverted Repeats and Target the Terminal Inverted Repeats of Tn21 Family Transposons

2003 ◽  
Vol 185 (21) ◽  
pp. 6371-6384 ◽  
Author(s):  
Sally R. Partridge ◽  
Ruth M. Hall
Keyword(s):  
Inverted Repeat ◽  
Insertion Sequence ◽  
Family Members ◽  
Target Site ◽  
Inverted Repeats ◽  
Terminal Inverted Repeats ◽  
Is Elements ◽  
Target Sites ◽  
Specific Position ◽  
The Right

ABSTRACT IS5075 and IS4321 are closely related (93.1% identical) members of the IS1111 family that target a specific position in the 38-bp terminal inverted repeats of Tn21 family transposons and that are inserted in only one orientation. They are 1,327 bp long and have identical ends consisting of short inverted repeats of 12 bp with an additional 7 bp (TAATGAG) or 6 bp (AATGAG) to the left of the left inverted repeats and 3 bp (AGA) or 4 bp (AGAT) to the right of the right inverted repeat. Circular forms of IS5075 and IS4321 in which the inverted repeats are separated by abutting terminal sequences (AGATAATGAG) were detected. A similar circular product was found for the related ISPa11. Transposition of IS4321 into the 38-bp target site was detected, but a flanking duplication was not generated. The precisely reconstituted target site was also identified. Over 50 members of the IS1111 family were identified. They encode related transposases, have related inverted repeats, and include related bases that lie outside these inverted repeats. In some, the flanking bases number 5 or 6 on the left and 4 or 3 on the right. Specific target sites were found for several of these insertion sequence (IS) elements. IS1111 family members therefore differ from the majority of IS elements, which are characterized by terminal inverted repeats and a target site duplication, and from members of the related IS110 family, which do not have obvious inverted repeats near their termini.

scholarly journals A systematic evaluation of data processing and problem formulation of CRISPR off-target site prediction

2021 ◽  
Author(s):  
Ofir Yaish ◽  
Maor Asif ◽  
Yaron Orenstein
Keyword(s):  
Data Processing ◽  
Gene Editing ◽  
Target Site ◽  
Systematic Evaluation ◽  
Guide Rna ◽  
Guide Rnas ◽  
Target Sites ◽  
Evaluation Of Data

AbstractCRISPR/Cas9 system is widely used in a broad range of gene-editing applications. While this gene-editing technique is quite accurate in the target region, there may be many unplanned off-target edited sites. Consequently, a plethora of computational methods have been developed to predict off-target cleavage sites given a guide RNA and a reference genome. However, these methods are based on small-scale datasets (only tens to hundreds of off-target sites) produced by experimental techniques to detect off-target sites with a low signal-to-noise ratio. Recently, CHANGE-seq, a new in vitro experimental technique to detect off-target sites, was used to produce a dataset of unprecedented scale and quality (more than 200,000 off-target sites over 110 guide RNAs). In addition, the same study included GUIDE-seq experiments for 58 of the guide RNAs to produce in vivo measurements of off-target sites. Here, we fill the gap in previous computational methods by utilizing these data to perform a systematic evaluation of data processing and formulation of the CRISPR off-target site prediction problem. Our evaluations show that data transformation as a pre-processing phase is critical prior to model training. Moreover, we demonstrate the improvement gained by adding potential inactive off-target sites to the training datasets. Furthermore, our results point to the importance of adding the number of mismatches between the guide RNA and the off-target site as a feature. Finally, we present predictive off-target in vivo models based on transfer learning from in vitro. Our conclusions will be instrumental to any future development of an off-target predictor based on high-throughput datasets.

scholarly journals Chemical control of a CRISPR-Cas9 acetyltransferase

2017 ◽  
Author(s):  
Jonathan H. Shrimp ◽  
Carissa Grose ◽  
Stephanie R. T. Widmeyer ◽  
Ajit Jadhav ◽  
Jordan L. Meier
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Critical Role ◽  
Regulation Of Transcription ◽  
Transcriptional Activators ◽  
Guide Rna ◽  
Transcriptional Reporter ◽  
Expression Control ◽  
Gene Expression Control ◽  
Spatiotemporal Control

AbstractLysine acetyltransferases (KATs) play a critical role in the regulation of transcription and other genomic functions. However, a persistent challenge is the development of assays capable of defining KAT activity directly in living cells. Towards this goal, here we report the application of a previously reported dCas9-p300 fusion as a transcriptional reporter of KAT activity. First we benchmark the activity of dCas9-p300 relative to other dCas9-based transcriptional activators, and demonstrate its compatibility with second generation short guide RNA architectures. Next, we repurpose this technology to rapidly identify small molecule inhibitors of acetylation-dependent gene expression. These studies validate a recently reported p300 inhibitor chemotype, and reveal a role for p300’s bromodomain in dCas9-p300-mediated transcriptional activation. Comparison with other CRISPR-Cas9 transcriptional activators highlights the inherent ligand tuneable nature of dCas9-p300 fusions, suggesting new opportunities for orthogonal gene expression control. Overall, our studies highlight dCas9-p300 as a powerful tool for studying gene expression mechanisms in which acetylation plays a causal role, and provide a foundation for future applications requiring spatiotemporal control over acetylation at specific genomic loci.

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