scholarly journals Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding

2019 ◽  
Author(s):  
David A. Specht ◽  
Yasu Xu ◽  
Guillaume Lambert
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Transcriptional Control ◽  
Target Recognition ◽  
Specific Binding ◽  
Massively Parallel ◽  
Nucleic Acid Detection ◽  
Factors Affecting ◽  
The Impact ◽  
Target Effects

The versatility of CRISPR-Cas endonucleases as a tool for biomedical research has lead to diverse applications in gene editing, programmable transcriptional control, and nucleic acid detection. Most CRISPR-Cas systems, however, suffer from off-target effects and unpredictable non-specific binding that negatively impact their reliability and broader applicability. To better evaluate the impact of mismatches on DNA target recognition and binding, we develop a massively parallel CRISPR interference (CRISPRi) assay to measure the binding energy between tens of thousands of CRISPR RNA (crRNA) and target DNA sequences. By developing a general thermodynamic model of CRISPR-Cas binding dynamics, our results unravel a comprehensive map of the energetic landscape of Francisella novicida Cas12a (FnCas12a) as it searches for its DNA target. Our results reveal concealed thermodynamic factors affecting FnCas12a DNA binding which should guide the design and optimization of crRNA that limit off-target effects, including the crucial role of an extended PAM sequence and the impact of the specific base composition of crRNA-DNA mismatches. Our generalizable approach should also provide a mechanistic understanding of target recognition and DNA binding when applied to other CRISPR-Cas systems.

scholarly journals Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding

2020 ◽  
Vol 117 (21) ◽  
pp. 11274-11282 ◽  
Author(s):  
David A. Specht ◽  
Yasu Xu ◽  
Guillaume Lambert
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Transcriptional Control ◽  
Target Recognition ◽  
Massively Parallel ◽  
Nucleic Acid Detection ◽  
Factors Affecting ◽  
Design And Optimization ◽  
The Impact ◽  
Target Effects

The versatility of CRISPR-Cas endonucleases as a tool for biomedical research has led to diverse applications in gene editing, programmable transcriptional control, and nucleic acid detection. Most CRISPR-Cas systems, however, suffer from off-target effects and unpredictable nonspecific binding that negatively impact their reliability and broader applicability. To better evaluate the impact of mismatches on DNA target recognition and binding, we develop a massively parallel CRISPR interference (CRISPRi) assay to measure the binding energy between tens of thousands of CRISPR RNA (crRNA) and target DNA sequences. By developing a general thermodynamic model of CRISPR-Cas binding dynamics, our results unravel a comprehensive map of the energetic landscape of nuclease-dead Cas12a (dCas12a) fromFrancisella novicidaas it inspects and binds to its DNA target. Our results reveal concealed thermodynamic factors affecting dCas12a DNA binding, which should guide the design and optimization of crRNA that limits off-target effects, including the crucial role of an extended protospacer adjacent motif (PAM) sequence and the impact of the specific base composition of crRNA–DNA mismatches. Our generalizable approach should also provide a mechanistic understanding of target recognition and DNA binding when applied to other CRISPR-Cas systems.

Making the cut(s): how Cas12a cleaves target and non-target DNA

2019 ◽  
Vol 47 (5) ◽  
pp. 1499-1510 ◽  
Author(s):  
Daan C. Swarts
Keyword(s):  
Conformational Changes ◽  
Dna Sequences ◽  
Dna Cleavage ◽  
Transcriptional Control ◽  
Molecular Mechanisms ◽  
Nucleic Acid Detection ◽  
Target Dna ◽  
Activated State ◽  
Dna Strand ◽  
Cis And Trans

Abstract CRISPR–Cas12a (previously named Cpf1) is a prokaryotic deoxyribonuclease that can be programmed with an RNA guide to target complementary DNA sequences. Upon binding of the target DNA, Cas12a induces a nick in each of the target DNA strands, yielding a double-stranded DNA break. In addition to inducing cis-cleavage of the targeted DNA, target DNA binding induces trans-cleavage of non-target DNA. As such, Cas12a–RNA guide complexes can provide sequence-specific immunity against invading nucleic acids such as bacteriophages and plasmids. Akin to CRISPR–Cas9, Cas12a has been repurposed as a genetic tool for programmable genome editing and transcriptional control in both prokaryotic and eukaryotic cells. In addition, its trans-cleavage activity has been applied for high-sensitivity nucleic acid detection. Despite the demonstrated value of Cas12a for these applications, the exact molecular mechanisms of both cis- and trans-cleavage of DNA were not completely understood. Recent studies have revealed mechanistic details of Cas12a-mediates DNA cleavage: base pairing of the RNA guide and the target DNA induces major conformational changes in Cas12a. These conformational changes render Cas12a in a catalytically activated state in which it acts as deoxyribonuclease. This deoxyribonuclease activity mediates cis-cleavage of the displaced target DNA strand first, and the RNA guide-bound target DNA strand second. As Cas12a remains in the catalytically activated state after cis-cleavage, it subsequently demonstrates trans-cleavage of non-target DNA. Here, I review the mechanistic details of Cas12a-mediated cis- and trans-cleavage of DNA. In addition, I discuss how bacteriophage-derived anti-CRISPR proteins can inhibit Cas12a activity.

scholarly journals Single position substitution of hairpin pyrrole-imidazole polyamides imparts distinct DNA-binding profiles across the human genome

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243905
Author(s):  
Paul B. Finn ◽  
Devesh Bhimsaria ◽  
Asfa Ali ◽  
Asuka Eguchi ◽  
Aseem Z. Ansari ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Dna Interaction ◽  
Sequence Specificity ◽  
Entire Genome ◽  
Dna Sequence Specificity ◽  
Interaction Sites ◽  
The Impact

Pyrrole–imidazole (Py–Im) polyamides are synthetic molecules that can be rationally designed to target specific DNA sequences to both disrupt and recruit transcriptional machinery. While in vitro binding has been extensively studied, in vivo effects are often difficult to predict using current models of DNA binding. Determining the impact of genomic architecture and the local chromatin landscape on polyamide-DNA sequence specificity remains an unresolved question that impedes their effective deployment in vivo. In this report we identified polyamide–DNA interaction sites across the entire genome, by covalently crosslinking and capturing these events in the nuclei of human LNCaP cells. This technique confirms the ability of two eight ring hairpin-polyamides, with similar architectures but differing at a single ring position (Py to Im), to retain in vitro specificities and display distinct genome-wide binding profiles.

scholarly journals Single position substitution of hairpin pyrrole-imidazole polyamides imparts distinct DNA-binding profiles across the human genome

2020 ◽  
Author(s):  
Paul B. Finn ◽  
Devesh Bhimsaria ◽  
Asfa Ali ◽  
Asuka Eguchi ◽  
Aseem Z. Ansari ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Dna Interaction ◽  
Sequence Specificity ◽  
Entire Genome ◽  
Dna Sequence Specificity ◽  
Interaction Sites ◽  
The Impact

ABSTRACTRegulating desired loci in the genome with sequence-specific DNA-binding molecules is a major goal for the development of precision medicine. Pyrrole–imidazole (Py–Im) polyamides are synthetic molecules that can be rationally designed to target specific DNA sequences to both disrupt and recruit transcriptional machinery. While in vitro binding has been extensively studied, in vivo effects are often difficult to predict using current models of DNA binding. Determining the impact of genomic architecture and the local chromatin landscape on polyamide-DNA sequence specificity remains an unresolved question that impedes their effective deployment in vivo. In this report we identified polyamide–DNA interaction sites across the entire genome, by covalently crosslinking and capturing these events in the nuclei of human LNCaP cells. This method, termed COSMIC-seq, confirms the ability of hairpin-polyamides, with similar architectures but differing at a single ring position, to retain in vitro specificities and display distinct genome-wide binding profiles. These results underpin the development of Py-Im polyamides as DNA-targeting molecules that mediate their regulatory or remedial functions at desired genomic loci.

scholarly journals Positive transcriptional control of the pyridoxal phosphate biosynthesis genes pdxST by the MocR-type regulator PdxR of Corynebacterium glutamicum ATCC 13032

Microbiology ◽  
2011 ◽  
Vol 157 (1) ◽  
pp. 77-88 ◽  
Author(s):  
Nina Jochmann ◽  
Susanne Götker ◽  
Andreas Tauch
Keyword(s):  
Dna Binding ◽  
Corynebacterium Glutamicum ◽  
Transcriptional Control ◽  
Pyridoxal Phosphate ◽  
Specific Binding ◽  
Consensus Sequence ◽  
Regulatory Protein ◽  
Band Shift ◽  
Promoter Sequences ◽  
Amino Terminal

The pdxR (cg0897) gene of Corynebacterium glutamicum ATCC 13032 encodes a regulatory protein belonging to the MocR subfamily of GntR-type transcription regulators and consisting of an amino-terminal winged helix–turn–helix DNA-binding domain and a carboxy-terminal aminotransferase-like domain. A defined deletion in the pdxR gene resulted in the decreased expression of the divergently orientated pdxST genes coding for the subunits of pyridoxal 5′-phosphate synthase. The pdxST mutant C. glutamicum NJ0898 and the pdxR mutant C. glutamicum AMH17 showed vitamin B6 auxotrophy that was restored by supplementing the growth medium with either pyridoxal, pyridoxal 5′-phosphate or pyridoxamine. The genetic organization of the 89 bp pdxR–pdxST intergenic region was elucidated by mapping the 5′ ends of the respective transcripts, followed by detection of typical promoter sequences. Bioinformatic pattern searches and comparative genomics revealed three DNA motifs with the consensus sequence AAAGTGGW(−/T)CTA, overlapping the deduced promoter sequences and serving as candidate DNA-binding sites for PdxR. DNA band shift assays with the purified PdxR protein demonstrated the specific binding of the transcription regulator to double-stranded 40-mer sequences containing the detected motifs, thereby confirming the direct regulatory role of PdxR in activating the expression of the pdxST genes.

scholarly journals Structural Insights into c-Myc-interacting Zinc Finger Protein-1 (Miz-1) Delineate Domains Required for DNA Scanning and Sequence-specific Binding

2016 ◽  
Vol 292 (8) ◽  
pp. 3323-3340 ◽  
Author(s):  
Mikaël Bédard ◽  
Vincent Roy ◽  
Martin Montagne ◽  
Pierre Lavigne
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Dna Sequences ◽  
Zinc Finger Protein ◽  
Specific Binding ◽  
Consensus Sequence ◽  
Compact Structure ◽  
General Role ◽  
Finger Protein

c-Myc-interacting zinc finger protein-1 (Miz-1) is a poly-Cys2His2 zinc finger (ZF) transcriptional regulator of many cell cycle genes. A Miz-1 DNA sequence consensus has recently been identified and has also unveiled Miz-1 functions in other cellular processes, underscoring its importance in the cell. Miz-1 contains 13 ZFs, but it is unknown why Miz-1 has so many ZFs and whether they recognize and bind DNA sequences in a typical fashion. Here, we used NMR to deduce the role of Miz-1 ZFs 1–4 in detecting the Miz-1 consensus sequence and preventing nonspecific DNA binding. In the construct containing the first 4 ZFs, we observed that ZFs 3 and 4 form an unusual compact and stable structure that restricts their motions. Disruption of this compact structure by an electrostatically mismatched A86K mutation profoundly affected the DNA binding properties of the WT construct. On the one hand, Miz1–4WT was found to bind the Miz-1 DNA consensus sequence weakly and through ZFs 1–3 only. On the other hand, the four ZFs in the structurally destabilized Miz1–4A86K mutant bound to the DNA consensus with a 30-fold increase in affinity (100 nm). The formation of such a thermodynamically stable but nonspecific complex is expected to slow down the rate of DNA scanning by Miz-1 during the search for its consensus sequence. Interestingly, we found that the motif stabilizing the compact structure between ZFs 3 and 4 is conserved and enriched in other long poly-ZF proteins. As discussed in detail, our findings support a general role of compact inter-ZF structures in minimizing the formation of off-target DNA complexes.

scholarly journals The regulatory function of dIno80 correlates with its DNA binding activity

2019 ◽  
Author(s):  
S Jain ◽  
J Maini ◽  
A Narang ◽  
S Maiti ◽  
V Brahmachari
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Dna Sequences ◽  
Specific Binding ◽  
Binding Domain ◽  
Regulatory Function ◽  
Binding Potential ◽  
Homeotic Genes ◽  
Dna Binding Domain

ABSTRACTThe INO80 complex, including the Ino80 protein, forms a highly conserved canonical complex that remodels chromatin in the context of multiple cellular functions. TheDrosophilahomologue, dIno80, is involved in homeotic gene regulation during development as a canonical Pho-dIno80 complex. Previously, we found that dIno80 regulates homeotic genes by interacting with epigenetic regulators, such as polycomb and trithorax, suggesting the occurrence of non-canonical Ino80 complexes. Here using spectroscopic methods and gel retardation assays, we identified a set of consensus DNA sequences that DNA binding domain of dIno80 (DBINO) interacts with having differential affinity and high specificity. Testing these sequences in reporter assays, showed that this interaction can positively regulate transcription. These results suggest that, dIno80 has a sequence preference for interaction with DNA leading to transcriptional changes.SIGNIFICANCEThe chromatin remodeling proteins control gene expression by nucleosome sliding and exchange. They are known to function as multi-subunit complexes recruited to chromatin by transcription factors or histone modification readers. Here, we report a sequence specific binding potential for the chromatin remodeler, dIno80. We have carried outin vitrostudies with DNA binding domain of dIno80 to elucidate its sequence specific DNA binding. We have also showed that this binding can regulated reporter gene expression inDrosophilacells. Our results suggest a non-canonical role of Ino80 in transcriptional regulation.

scholarly journals Which Factors Affect the Occurrence of Off-Target Effects Caused by the Use of CRISPR/Cas: A Systematic Review in Plants

2020 ◽  
Vol 11 ◽  
Author(s):  
Dominik Modrzejewski ◽  
Frank Hartung ◽  
Heike Lehnert ◽  
Thorben Sprink ◽  
Christian Kohl ◽  
...  
Keyword(s):  
Systematic Review ◽  
Dna Sequences ◽  
Research Question ◽  
Gc Content ◽  
Binary Logistic Regression ◽  
Binary Logistic Regression Analysis ◽  
Target Sequence ◽  
Target Sequences ◽  
The Impact ◽  
Target Effects

CRISPR/Cas enables a targeted modification of DNA sequences. Despite their ease and efficient use, one limitation is the potential occurrence of associated off-target effects. This systematic review aims to answer the following research question: Which factors affect the occurrence of off-target effects caused by the use of CRISPR/Cas in plants? Literature published until March 2019 was considered for this review. Articles were screened for relevance based on pre-defined inclusion criteria. Relevant studies were subject to critical appraisal. All studies included in the systematic review were synthesized in a narrative report, but studies rated as high and medium/high validity were reported separately from studies rated as low and medium/low or unclear validity. In addition, we ran a binary logistic regression analysis to verify five factors that may affect the occurrence of off-target effects: (1) Number of mismatches (2) Position of mismatches (3) GC-content of the targeting sequence (4) Altered nuclease variants (5) Delivery methods. In total, 180 relevant articles were included in this review containing 468 studies therein. Seventy nine percentage of these studies were rated as having high or medium/high validity. Within these studies, 6,416 potential off-target sequences were assessed for the occurrence of off-target effects. Results clearly indicate that an increased number of mismatches between the on-target and potential off-target sequence steeply decreases the likelihood of off-target effects. The observed rate of off-target effects decreased from 59% when there is one mismatch between the on-target and off-target sequences toward 0% when four or more mismatches exist. In addition, mismatch/es located within the first eight nucleotides proximal to the PAM significantly decreased the occurrence of off-target effects. There is no evidence that the GC-content significantly affects off-target effects. The database regarding the impact of the nuclease variant and the delivery method is very poor as the majority of studies applied the standard nuclease SpCas9 and the CRISPR/Cas system was stably delivered in the genome. Hence, a general significant impact of these two factors on the occurrence of off-target effects cannot be proved. This identified evidence gap needs to be filled by systematic studies exploring these individual factors in sufficient numbers.

scholarly journals Actinomycin D specifically inhibits the interaction between transcription factor Sp1 and its binding site.

1998 ◽  
Vol 45 (1) ◽  
pp. 67-73 ◽  
Author(s):  
M Czyz ◽  
M Gniazdowski
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Dna Sequences ◽  
Actinomycin D ◽  
Protein Complexes ◽  
Specific Binding ◽  
Umbilical Vein ◽  
Nuclear Proteins ◽  
Mobility Shift ◽  
Umbilical Vein Endothelial Cells

The mode of action of many anticancer drugs involves DNA interactions. We here examine the ability of actinomycin D to alter the specific binding of transcription factors Spl and NFkappaB to their DNA sequences. Employing an electrophoretic mobility shift assay, it is shown that actinomycin D inhibits complex formation between nuclear proteins present in the extracts from stimulated human umbilical vein endothelial cells and the Sp1-binding site. Actinomycin D is also able to induce disruption of preformed DNA-protein complexes, pointing to the importance of an equilibrium of three components: actinomycin D, protein and DNA for drug action. The effect of actinomycin D is sequence-specific, since no inhibition is observed for interaction of nuclear proteins with the NFkappaB binding site. The results support the view that DNA-binding drugs displaying high sequence-selectivity can exhibit distinct effects on the interaction between DNA and different DNA-binding proteins.

DAMPAK KEBIJAKAN FISKAL TERHADAP KINERJA SEKTOR PERTANIAN DI SULAWESI TENGGARA

2017 ◽  
Vol 1 (1) ◽  
Author(s):  
La Ode Jabuddin ◽  
Ayub M Padangaran ◽  
Azhar Bafadal Bafadal
Keyword(s):  
Fiscal Policy ◽  
Interest Rates ◽  
Agricultural Sector ◽  
Simultaneous Equations ◽  
Poor People ◽  
Land Area ◽  
Factors Affecting ◽  
Historical Simulation ◽  
The Impact ◽  
Direct Expenditure

This study aims to: (1) Knowing the dynamics of fiscal policy and the performance of the agricultural sector, (2) Analyze the factors that influence fiscal policy and the performance                   of the agricultural sector, and (3) Analyzing the impact of fiscal policy on the performance of the agricultural sector. The data used in this study were pooled 2005-2013 data in the aggregate. Econometric model the impact of fiscal policy on the performance of the agricultural sector is built in the form of simultaneous equations, consisting of 7 equations with 25 total variables in the model, 7 endogenous variables, 12 exogenous variables, and 6 variables lag. The model is estimated by 2SLS method SYSLIN procedures and historical simulation with SIMNLIN procedure.The results showed that: (1) The development of fiscal policy in Southeast Sulawesi from year to year tends to increase, (2) The performance of the agricultural sector from the aspect of GDP has decreased, from the aspect of labor is still consistent, in terms of investment to grow positively, and assign roles which means to decrease the number of poor people, (3) factors affecting fiscal policy is local revenues, equalization funds, other revenues, as well as the lag fiscal policy, (4) the factors that affect the performance of the agricultural sector from the aspect GDP is labor, direct expenditure and GDP lag; from the aspect of labor is the total labor force, investment, land area, direct expenditure, as well as the lag of labor; from the aspect of investment is influenced by GDP per capita, land area, interest rates and investment lag; as well as from the aspect of poor people, are affected by population, investments, direct expenditure and poverty lag, (5). Fiscal policy impact on the agricultural sector GDP increase, a decrease in the number of poor, declining agricultural laborers, and a decrease in the amount of investment in the agricultural sector.Keywords: Fiscal policy, the performance of the agricultural sector, the simultaneous equations

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