scholarly journals Phosphorothioate-modified DNA oligonucleotides inactivate CRISPR-Cpf1 mediated genome editing

2018 ◽  
Author(s):  
Bin Li ◽  
Chunxi Zeng ◽  
Wenqing Li ◽  
Xinfu Zhang ◽  
Xiao Luo ◽  
...  
Keyword(s):  
Genome Editing ◽  
Adaptive Immune System ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Dna Oligonucleotides ◽  
Double Stranded Dna ◽  
Modified Dna ◽  
Target Dna ◽  
Editing Activity ◽  
Dose Dependent

CRISPR-Cpf1, a microbial adaptive immune system discovered from Prevotella and Francisella 1, employs a single-stranded CRISPR RNA (crRNA) to induce double stranded DNA breaks1. To modulate genome editing activity of Cpf1 in human cells, we designed a series of crRNA variants including DNA-crRNA and RNA-crRNA duplexes, and identified that phosphorothioate (PS)-modified DNA-crRNA duplex completely blocked the function of Cpf1 mediated gene editing. More importantly, without prehybridization, this PS-modified DNA was able to regulate Cpf1 activity in a time-and dose-dependent manner. Mechanistic studies indicate that PS-modified DNA oligonucleotides hinder the binding between Cpf1-crRNA complex and target DNA substrate. Consequently, phosphorothioate-modified DNA oligonucleotides provide a tunable platform to inactivate Cpf1 mediated genome editing.

scholarly journals Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Zhiwei Hu ◽  
Yannan Wang ◽  
Qian Liu ◽  
Yan Qiu ◽  
Zhiyu Zhong ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Breaks ◽  
Single Nucleotide ◽  
Base Editing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Double Stranded Dna ◽  
Target Sites ◽  
Guide Sequence ◽  
Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

scholarly journals Mechanistic Insights into theCis-andTrans-acting Deoxyribonuclease Activities of Cas12a

2018 ◽  
Author(s):  
Daan C. Swarts ◽  
Martin Jinek
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Cleavage Product ◽  
List Type ◽  
Francisella Novicida ◽  
Ssdna Binding ◽  
Double Stranded Dna ◽  
Target Dna ◽  
Dna Strands ◽  
Dna Strand

HIGHLIGHTSTarget ssDNA binding allosterically induces unblocking of the RuvC active sitePAM binding facilitates unwinding of dsDNA targetsNon-target DNA strand cleavage is prerequisite for target DNA strand cleavageAfter DNA cleavage, Cas12a releases the PAM-distal DNA productSUMMARYCRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA incisand non-target single stranded DNAs (ssDNA) intrans.To elucidate the molecular basis for both deoxyribonuclease cleavage modes, we performed structural and biochemical studies onFrancisella novicidaCas12a. We show how crRNA-target DNA strand hybridization conformationally activates Cas12a, triggering itstrans-acting, non-specific, single-stranded deoxyribonuclease activity. In turn,cis-cleavage of double-stranded DNA targets is a result of PAM-dependent DNA duplex unwinding and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent,trans-active state. Together, these results provide a revised model for the molecular mechanism of Cas12a enzymes that explains theircis- andtrans-acting deoxyribonuclease activities, and additionally contribute to improving Cas12a-based genome editing.

scholarly journals D-Alanyl-D-Alanine Ligase as a Broad-Host-Range Counterselection Marker in Vancomycin-Resistant Lactic Acid Bacteria

2018 ◽  
Vol 200 (13) ◽  
Author(s):  
Shenwei Zhang ◽  
Jee-Hwan Oh ◽  
Laura M. Alexander ◽  
Mustafa Özçam ◽  
Jan-Peter van Pijkeren
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Heterologous Expression ◽  
Genome Editing ◽  
Vancomycin Resistance ◽  
Dependent Manner ◽  
Content Type ◽  
Suicide Vector ◽  
Vancomycin Resistant ◽  
Dose Dependent

ABSTRACTThe peptidoglycan composition in lactic acid bacteria dictates vancomycin resistance. Vancomycin binds relatively poorly to peptidoglycan ending ind-alanyl-d-lactate and binds with high affinity to peptidoglycan ending ind-alanyl-d-alanine (d-Ala-d-Ala), which results in vancomycin resistance and sensitivity, respectively. The enzyme responsible for generating these peptidoglycan precursors is dipeptide ligase (Ddl). A single amino acid in the Ddl active site, phenylalanine or tyrosine, determines depsipeptide or dipeptide activity, respectively. Here, we established that heterologous expression of dipeptide ligase in vancomycin-resistant lactobacilli increases their sensitivity to vancomycin in a dose-dependent manner and overcomes the effects of the presence of a natived-Ala-d-Ala dipeptidase. We incorporated the dipeptide ligase gene on a suicide vector and demonstrated that it functions as a counterselection marker (CSM) in lactobacilli; vancomycin selection allows only those cells to grow in which the suicide vector has been lost. Subsequently, we developed a liquid-based approach to identify recombinants in only 5 days, which is approximately half the time required by conventional approaches. Phylogenetic analysis revealed that Ddl serves as a marker to predict vancomycin resistance and consequently indicated the broad applicability of the use of Ddl as a counterselection marker in the genusLactobacillus. Finally, our system represents the first “plug and play” counterselection system in lactic acid bacteria that does not require prior genome editing and/or synthetic medium.IMPORTANCEThe genusLactobacilluscontains more than 200 species, many of which are exploited in the food and biotechnology industries and in medicine. Prediction of intrinsic vancomycin resistance has thus far been limited to selectedLactobacillusspecies. Here, we show that heterologous expression of the enzyme Ddl (dipeptide ligase)—an essential enzyme involved in peptidoglycan synthesis—increases sensitivity to vancomycin in a dose-dependent manner. We exploited this to develop a counterselection marker for use in vancomycin-resistant lactobacilli, thereby expanding the poorly developed genome editing toolbox that is currently available for most strains. Also, we showed that Ddl is a phylogenetic marker that can be used to predict vancomycin resistance inLactobacillus; 81% ofLactobacillusspecies are intrinsically resistant to vancomycin, which makes our tool broadly applicable.

scholarly journals Mre11-Rad50-Nbs1 complex is activated by hypertonicity

2006 ◽  
Vol 291 (5) ◽  
pp. F1014-F1020 ◽  
Author(s):  
Mee Rie Sheen ◽  
Seung Whan Kim ◽  
Ju-Young Jung ◽  
Joon Young Ahn ◽  
Juong G. Rhee ◽  
...  
Keyword(s):  
Cellular Response ◽  
Control Level ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Dna Damages ◽  
Mrn Complex ◽  
Double Strand Dna Breaks ◽  
Functionally Impaired ◽  
Mutant Cells ◽  
Dose Dependent

When exposed to hypertonic conditions, cells accumulate double-strand DNA breaks (DSBs) like they are exposed to ionizing radiation. It has been proposed that inactivation of the Mre11-Rad50-Nbs1 (MRN) complex due to nuclear exit is responsible for the accumulation of DSBs as cells fail to repair DSBs produced during normal cellular activity. In this study, we examined the MRN complex in cells switched to hypertonicity. Surprisingly, we found that the MRN complex stayed in the nucleus and remained intact in response to hypertonicity. In fact, the MRN complex was dramatically activated after 4 h of switch to hypertonicity in a dose-dependent manner as shown by formation of foci. Activation of ATM and the MRN complex by hypertonicity and bleomycin was additive as was activation of their downstream targets including γH2AX and Chk2 indicating that the cellular response to DSB was intact in hypertonic conditions. Activation of Chk2 in response to hypertonicity was not observed in mutant cells with functionally impaired MRN complex confirming that they are in the same pathway. After 20 h of a switch to hypertonicity, MRN foci and γH2AX returned to a control level, suggesting that cells adapted to hypertonicity by repairing DNA. We conclude that cells respond normally to DSB and repair the DNA damages induced by hypertonicity.

scholarly journals Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break

2019 ◽  
Author(s):  
Geoffrey L. Rogers ◽  
Hsu-Yu Chen ◽  
Heidy Morales ◽  
Paula M. Cannon
Keyword(s):  
Homologous Recombination ◽  
Progenitor Cells ◽  
Genome Editing ◽  
High Efficiency ◽  
Zinc Finger Nucleases ◽  
Dna Breaks ◽  
Adeno Associated Virus ◽  
Hematopoietic Stem ◽  
Double Stranded Dna ◽  
Dna Break

AbstractAdeno-associated virus (AAV) vectors are frequently used as donor templates for genome editing by homologous recombination. Although modification rates are typically under 1%, they are greatly enhanced by targeted double-stranded DNA breaks (DSBs). A recent report described clade F AAVs mediating high-efficiency homologous recombination-based editing in the absence of DSBs. The clade F vectors included AAV9 and a series isolated from human hematopoietic stem/progenitor cells (HSPCs). We evaluated these vectors by packaging homology donors into AAV9 and an AAVHSC capsid and examining their ability to insert GFP at the CCR5 or AAVS1 loci in human HSPCs and cell lines. As a control we used AAV6, which effectively edits HSPCs, but only when combined with a targeted DSB. Each AAV vector promoted GFP insertion in the presence of matched CCR5 or AAVS1 zinc finger nucleases (ZFNs), but none supported detectable editing in the absence of the nucleases. Rates of editing with ZFNs correlated with transduction efficiencies for each vector, implying no differences in the ability of donor sequences delivered by the different vectors to direct genome editing. Our results therefore do not support that clade F AAVs can perform high efficiency genome editing in the absence of a DSB.

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 PAM recognition by miniature CRISPR–Cas12f nucleases triggers programmable double-stranded DNA target cleavage

2020 ◽  
Vol 48 (9) ◽  
pp. 5016-5023 ◽  
Author(s):  
Tautvydas Karvelis ◽  
Greta Bigelyte ◽  
Joshua K Young ◽  
Zhenglin Hou ◽  
Rimante Zedaveinyte ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Genome Editing ◽  
Dependent Manner ◽  
Short Sequence ◽  
Double Stranded Dna ◽  
Protospacer Adjacent Motif ◽  
Type V ◽  
Programmable Nucleases ◽  
Cas Proteins

Abstract In recent years, CRISPR-associated (Cas) nucleases have revolutionized the genome editing field. Being guided by an RNA to cleave double-stranded (ds) DNA targets near a short sequence termed a protospacer adjacent motif (PAM), Cas9 and Cas12 offer unprecedented flexibility, however, more compact versions would simplify delivery and extend application. Here, we present a collection of 10 exceptionally compact (422–603 amino acids) CRISPR–Cas12f nucleases that recognize and cleave dsDNA in a PAM dependent manner. Categorized as class 2 type V-F, they originate from the previously identified Cas14 family and distantly related type V-U3 Cas proteins found in bacteria. Using biochemical methods, we demonstrate that a 5′ T- or C-rich PAM sequence triggers dsDNA target cleavage. Based on this discovery, we evaluated whether they can protect against invading dsDNA in Escherichia coli and find that some but not all can. Altogether, our findings show that miniature Cas12f nucleases can protect against invading dsDNA like much larger class 2 CRISPR effectors and have the potential to be harnessed as programmable nucleases for genome editing.

scholarly journals PAM recognition by miniature CRISPR nucleases triggers programmable double-stranded DNA target cleavage

2019 ◽  
Author(s):  
Tautvydas Karvelis ◽  
Greta Bigelyte ◽  
Joshua K. Young ◽  
Zhenglin Hou ◽  
Rimante Zedaveinyte ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dependent Manner ◽  
Short Sequence ◽  
E Coli ◽  
Defense Systems ◽  
Double Stranded Dna ◽  
Protospacer Adjacent Motif ◽  
Type V ◽  
Programmable Nucleases ◽  
Cas Proteins

ABSTRACTIn recent years, CRISPR-associated (Cas) nucleases have revolutionized the genome editing field. Being guided by an RNA to cleave double-stranded (ds) DNA targets near a short sequence termed a protospacer adjacent motif (PAM), Cas9 and Cas12 offer unprecedented flexibility, however, more compact versions would simplify delivery and extend application. Here, we present a collection of 10 exceptionally compact (422-603 amino acids) CRISPR-Cas nucleases that recognize and cleave dsDNA in PAM dependent manner. Categorized as class 2 type V-F they come from the Cas14 family and distantly related type V-U3 Cas proteins found in bacteria. Using biochemical methods, we demonstrate that a 5’ T- or C-rich PAM sequence triggers double stranded (ds) DNA target cleavage. Based on this discovery, we evaluated whether they can protect against invading dsDNA in E. coli and find that some but not all can. Altogether, our findings show that miniature Cas nucleases are functional CRISPR-Cas defense systems and have the potential to be harnessed as programmable nucleases for genome editing.

Label-free detection of double-stranded DNA molecules with polyelectrolyte-modified capacitive field-effect sensors

2017 ◽  
Vol 84 (10) ◽  
Author(s):  
Thomas S. Bronder ◽  
Arshak Poghossian ◽  
Michael Keusgen ◽  
Michael J. Schöning
Keyword(s):  
Field Effect ◽  
Deoxyribonucleic Acid ◽  
Label Free ◽  
Electrical Detection ◽  
Dna Oligonucleotides ◽  
Double Stranded Dna ◽  
Polyelectrolyte Layer ◽  
Target Dna ◽  
Label Free Detection ◽  
Positively Charged

AbstractIn this study, polyelectrolyte-modified field-effect-based electrolyte-insulator-semiconductor (EIS) devices have been used for the label-free electrical detection of double-stranded deoxyribonucleic acid (dsDNA) molecules. The sensor-chip functionalization with a positively charged polyelectrolyte layer provides the possibility of direct adsorptive binding of negatively charged target DNA oligonucleotides onto the SiO

scholarly journals Effects of a thyromimetic on apolipoprotein B-100 in rats

2000 ◽  
Vol 25 (3) ◽  
pp. 299-308 ◽  
Author(s):  
Y Wada ◽  
S Matsubara ◽  
J Dufresne ◽  
GM Hargrove ◽  
ZF Stephan ◽  
...  
Keyword(s):  
Apolipoprotein B ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Mrna Editing ◽  
Human Hepatoma Cells ◽  
Apo B ◽  
Editing Activity ◽  
Cardiac Sparing ◽  
Dose Dependent

We have studied the effects of a cardiac sparing thyromimetic, CGS 23425, on postprandial levels of triglycerides, abundance of apolipoprotein B (apo B) protein and hepatic apo B mRNA expression in rats. When compared with control rats, triglyceride clearance was significantly accelerated by treatment with CGS 23425. A full return to baseline values was achieved within 8 h after ingesting a large quantity of fat, as compared to >24 h in control animals. The abundance of apo B-100 protein in CGS 23425-treated hyperlipidemic rats decreased in a dose-dependent manner, but levels of apo B-48 were not significantly affected. Like L-tri-iodothyronine (L-T(3)), treatment with 30 microg/kg CGS 23425 for 6 or 9 days decreased the levels of apo B-100 protein by 80% and 40% respectively. This change was paralleled by a 27% reduction in hepatic apo B-100 mRNA. To investigate a potential mechanism of CGS 23425 action, we measured in vitro apo B mRNA editing activity in hepatocellular extract from control or CGS 23425-treated rats. Treatment with CGS 23425 increased activity of the hepatic apo B-100 editosome, apobec-1. In human hepatoma cells which lack apobec-1 activity, apo B-100 mRNA levels remained the same in cells treated with or without the agent. In summary, these observations show that CGS 23425 decreases the levels of apo B-100 in rats. This action of CGS 23425 involves apo B-100 mRNA editing activity.

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