...MEDLINE-indexed journal Nuclear Receptor Signaling. Dr. McKenna edits the website of the Nuclear Receptor Signaling... elicit their actions on other tissues. At the Nuclear Receptor Signaling Atlas (NURSA), one of our primary...regulated at the cellular, tissue and organ level by nuclear receptors (NRs), their coregulators, and their ...their physiological endocrine ligands. Nuclear receptors and their ligands NRs represent the largest family...Consortium represents a unique funding model in nuclear receptor research, and has generated a set of comprehensive...address many important and fundamental questions in nuclear receptor and coregulator research. Our Q-PCR datasets...for example, document the expression patterns of nuclear receptors across multiple different tissues in ...

... 4D nucleome, the organization of the nucleus in space and time, and to understanding how nuclear organization...dCas9 gets a colorful makeover Even without its nuclease activity, catalytically dead Cas9 (dCas9) has ...loci, a major benchmark in characterizing the 4D nucleome. Future modifications and applications Ma et al...technique. The NIH Common Fund has made mapping the 4D nucleome a specific priority, as researchers work to increase... nucleome...

...updated April 16, 2020. Sensitive and specific nucleic acid detection is crucial for clinical diagnostics...biotechnological advancements. Many methods of nucleic acid detection however, either lack the sensitivity...sensitivity or the specificity to detect nucleic acids at low concentrations and/or are too expensive, time-consuming..., and inexpensive platforms to reliably detect nucleic acids at the atomolar level. The Zhang lab has...degradation to develop the method termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) (Chen...signal that indicates the presence of your targeted nucleic acid. To increase the sensitivity of the assay,... In addition, Csm6, a CRISPR type-III effector nuclease, can be used in conjunction with Cas13 to amplify...

...reagents in a technique known as nucleofection, which forms pores in the nuclear membrane, allowing for entry...translation delays editing What are Cas9-gRNA ribonucleoproteins? One alternative approach, which avoids many...these complications, is to directly deliver a ribonucleoprotein (RNP), consisting of the Cas9 protein in complex...use RNPs, now what? Preparing the Cas9-gRNA ribonucleoprotein The first step in this type of CRISPR experiment...Schier laboratories. Delivering Cas9-gRNA ribonucleoproteins  An advantage of performing CRISPR experiments...fusion to a fluorescent protein is used to label nucleic acid targets.  While Cas9-gRNA RNPs show promise... efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes." Journal of Biological...

...Considerations when choosing your Cas nuclease When choosing the Cas9 nuclease for your experiments, there are...but since then, scientists have adapted CRISPR nucleases for many more specific research needs. In this...determined by the PAM sequence recognized by the nuclease. For the most frequently used Cas9, spCas9 from... Figure 1: The PAM sequence that the Cas nuclease recognizes determines site accessibility. Cas9s... measure of the on-target activity of the Cas9 nuclease. This can be determined by measuring the indel...measure of on-target activity (right). Cas9 nucleases that exhibit enhanced site accessibility In order...engineered Cas9, SpCas9-NG, which required only the di-nucleotide PAM NG (Nishimasu et al., 2018). The David Liu...

...time using Custom Synthetic cr:tracrRNA/Cas9 Ribonucleoprotein (csRNP) delivery Epitope tagging of mammalian...with recombinant Cas9 protein to form the ribonucleoprotein complex (csRNP, Figure 1). We use in-house...cells recover for 2-5 days.Note: We use the Nucleofection method for delivery of csRNP reagents into cells...tagging in mammalian stem cells using Cas9 ribonucleoprotein. Elife 7. PubMed PMID: 29638216. PubMed Central... by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA. J Biotechnol 241: 136-146. PubMed...

... participate when the organizers of the Canton Nucleic Acids Forum (CNAF) 2015 conference invited me to...Chinese scientists eager to hear current findings in nucleic acid science. I was delighted to see that the majority...plasmid requests from Addgene. Addgene - Canton Nucleic Acids Forum 2015 from Joanne Kamens, PhD Thoughts...structure.” That certainly summed up this conference—nucleic acid science still has many surprises for the researchers... nucleic acids...

...two major lobes, called NUC (for nuclease) and REC (for recognition). The NUC lobe contains the DNA scissors... of Cas9’s two major lobes, REC and NUC, and their subdomains. NUC includes the HNH and RuvC catalytic...conformation cleaves both DNA strands via the NUC lobe, as in Figure 6. NUC lobe, green; REC lobe, light blue; guide... domain (CTD) of the protein also belongs to the NUC lobe. Because its primary role is recognizing the...to, and their roles. Lobe Domain Role NUC HNH Cut DNA target strand RuvC Cut DNA ...’s most often shown in an “open” state, with the NUC and REC lobes laying open like a book (Figure 2)....conformation, as in Figure 3. Upon DNA binding, the NUC lobe recognizes the PAM site in the non-target DNA...

...pAAV-mDlx-NLS-mRuby2 112677 AAV1, AAV2 pOTTC1032 - pAAV EF1a Nuc-flox(mCherry)-EGFP 27056 AAVrg pAAV-Ef1a-DIO EYFP...pAAV-Ef1a-fDIO mCherry 112677 AAV2 pOTTC1032 - pAAV EF1a Nuc-flox(mCherry)-EGFP 50457 AAV2 pAAV-hSyn-DIO-EGFP...

...forebrain GABAergic interneurons. pOTTC1032 - pAAV EF1a Nuc-flox(mCherry)-EGFP (112677-AAV5 and 112677-AAVrg)...H134R)-mCherry (26976-AAV5) pOTTC1032 - pAAV EF1a Nuc-flox(mCherry)-EGFP (112677-AAV5) Calcium Sensors...switch that expresses nuclear mCherry in the absence of Cre, and expresses nuclear EGFP in the presence...

...Karl Deisseroth 112677 AAV2 pOTTC1032 - pAAV EF1a Nuc-flox(mCherry)-EGFP Brandon Harvey 50457 AAV2 pAAV-hSyn-DIO-EGFP...

...mammalian cells. Like Cas9 nucleases, Cpf1 family members contain a RuvC-like endonuclease domain, but they lack...contains a RuvC-like endonuclease domain, but it lacks Cas9’s other HNH endonuclease domain, indicating ...functional CRISPR nuclease that could be adapted for genome editing. The use of a different nuclease could potentially...lack Cas9’s second HNH endonuclease domain. Cpf1 cleaves DNA in a staggered pattern and requires only ... Figure 1: Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Figure from ... Cas9-based system, require a single-component nuclease to mediate cleavage rather than the multi-subunit...employed by class 1 systems. A putative new class 2 nuclease, Cpf1 (CRISPR from Prevotella and Francisella)...

...mutated to inactivate the protein’s nucleolytic capability, and this nuclease-null or “dead” Cas9 (dCas9) variant...relies on two distinct Cas9 variants—nuclease-competent or nuclease-null—in order to effect different perturbations...imagine transfecting cells with both the nuclease-positive and nuclease-null variants of Cas9 along with the...(gRNA). The gRNA conventionally contains a 20-nucleotide sequence that is complementary to the target ...Alex Chavez Figure 1: Schematic of native (nuclease-competent) Cas9 interacting with either a full-length... many cases this is due both to lower relative nuclease efficiencies as well as to more stringent targeting... An alternative approach is to modulate Cas9’s nuclease activity by modifying the gRNA with which it is...

...serve as slaves.  Meganucleases In 1989 the first ever DNA-editing enzymes, meganucleases, were discovered...created by genetics.    Zinc finger nucleases Zinc finger nucleases (ZFNs) are artificial restriction enzymes...instead of recognizing nucleotide triplets, the components recognize individual nucleotides. This imparts TALENs...The major drawback of all the endonuclease-based gene editors (meganucleases, ZFNs, TALENs, and CRISPR-Cas...altering the cleavage specificity of a homing endonuclease. Nucleic Acids Research 30:3870–3879 . https://doi.org...in 1990, just a year after the discovery of meganucleases. The storyline involves the resurrection of ... to a DNA-cleavage domain (FokI). Zinc finger nucleases were first designed in 2005. The DNA-binding domain...

..."The polynucleotide ligase and RNA capping enzyme superfamily of covalent nucleotidyltransferases." Current...mispaired bases, and one-nucleotide gaps can be sealed by T4 DNA ligase." Nucleic acids research 15.21 (...al. "Ligase-based detection of mononucleotide repeat sequences." Nucleic acids research 27.24 (1999): e40...accelerate characterization of nucleic acid metabolic enzymes." Nucleic acids research 44.2 (2015): e15...DNA ligases. These applications detect specific nucleotide sequences with high specificity and quantitative...quantitative accuracy and even include profiling single nucleotide polymorphisms (SNPs) (5,12,13). For example, ...David H. Gelfand. "Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding...

...that occurs during nuclear envelope breakdown (without the nucleus, there’s no nuclear localization to monitor...further fused to a bipartite nuclear localization signal (bNLS) and a nuclear export signal (NES), both ...the nucleus depending on its phosphorylation status. The relative ratio of cytoplasmic vs. nuclear fluorescence... because KTRs shuttle between the nucleus and the cytoplasm, nuclear export and import rates can influence...Prior to phosphorylation, the KTR localizes to the nucleus, but following phosphorylation bNLS activity is...kinase activity that occurs in the cytoplasm and/or nucleus, while FRET-based reporters can be used to measure... rates have negligible effect on KTRs, however nuclear translocation rates are another variable to consider...

...Transformed cell lines Cargo Nucleic acids or protein Nucleic acids Nucleic acids Throughput High High... Dividing or nondividing Cargo Nucleic acids Nucleic acids Nucleic acids Throughput Low Low Low ...Electroporation / Nucleofection Microinjection Cells Many types Single cells Cargo Nucleic acids or protein...deliver a donor plasmid or single-stranded DNA oligonucleotide, so you will need to make sure your expression...reagents can be delivered via transfection, nucleofection, viral transduction or injection as either protein...generating transgenic embryos. Cas9-gRNA ribonucleoprotein complexes Purified Cas9 protein and in vitro...protein Nucleic acids or protein Throughput High Very low Efficiency High Very high Difficulty Very...

...have two endonuclease domains: the n-terminal RuvC-like nuclease domain and the HNH-like nuclease domain...Cas9 genome editing systems use a non-specific endonuclease (Cas9 or closely related Cpf1) to cut the genome...genome and a small RNA (gRNA) to guide this nuclease to a user-defined cut site. After reading this post... have increased targeting specificity). Cas9 endonuclease functions While native CRISPR/Cas systems have...foreign DNA as well as the RNA guides required for endonuclease function, when used for genome engineering, ...the only CRISPR protein required is the Cas9 endonuclease or a variant thereof. This individual protein...) of a protospacer adjacent motif (PAM) Cas9 endonuclease binding to the target genomic locus is mediated...

...vulnerable to attack by both cytoplasmic and nuclear nucleases, as they will be recognized as foreign RNA...to simply swap out several of the ribonucleotides for deoxynucleotides. A partially DNA gRNA is surprisingly...efficiency. In the same vein, locked nucleic acids or bridged nucleic acids can also be introduced in the...hydroxyl group of the ribose to protect it from nucleolytic attack. Another stabilizing sugar modification...modifications to consider include amide linkages, unlocked nucleic acids, and a constrained ethyl.  Can’t decide which...which improves mismatch discrimination and is more nuclease-resistant, resulting in less off-target events...events. While both have similar advantages, bridged nucleic acids with N-methyl substitutions are slightly ...

...TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5. Nucleic Acids Research...researchers instead turn to so-called “mismatch nuclease” assays (e.g. Surveyor® or T7E1) for mutation ...Digital PCR has several advantages over mismatch nuclease assays that will be elaborated below. Digital...advantages over the commonly employed mismatch nuclease assays. Sensitivity: Less than 20 ng of total...mutant targets have been reported for mismatch nuclease assays (2, 6, 7). Ability to distinguish between...functional knockout of your target gene. Mismatch nuclease assays are completely “blind” to this very important...gene-editing frequencies (GEF-dPCR) mediated by designer nucleases. Nat Protoc 11, 598–615 (2016). PubMed PMID: 26914317...