...nuclease specificity using truncated guide RNAs. 2014. Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. Nat Biotechnol...induced by CRISPR-Cas nucleases in human cells. 2013. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung...also be fused with transcriptional repressors or activators, and targeting these dCas9 fusion proteins...increase HDR frequency. Additionally, Cas9-CtIP , a fusion of Cas9 and CtIP, a protein involved in double-...editors (ABEs) . Cytosine base editors are created by fusing Cas9 nickase or catalytically inactive “dead” Cas9...cytosine base editors, the evolved TadA domain is fused to a Cas9 protein to create the adenine base editor...have been made by optimizing expression of the fusions, modifying the linker region between Cas variant...

...Human Cells. Cell Rep . 13(3):621-33. PMID: 26456817 Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. 2014....convert adenosine to guanosine. RNA editors : Cas13 fused to the ADAR2 deaminase convert adenosine to inosine...modifications at specific loci. Activate or Repress : dCas9 fused to transcriptional activators or repressors can ...transcription in a reversible manner. Epigenetics : dCas9 fused to epigenetic modifiers can modulate transcriptions...

...frame with tags or fusion proteins in the recipient plasmid (if you are creating a fusion protein) Ideally...insert into your vector: Conduct a DNA Ligation to fuse your insert to your recipient plasmid. We recommend...

...the two pieces of DNA connect and ultimately are fused by the ligation reaction. The example below depicts...therefore prevents the ligase from being able to fuse the two ends of the vector together). Protocol: ...

...and the bottle contents. This will clear up any confusion later if your forget your bottle in the autoclave...likely that you’ve used the wrong antibiotic, or confused your strains....

...in the recipient plasmid by viewing the MCS and fusing the upstream restriction site to the forward primer...insert into your vector: Conduct a DNA Ligation to fuse your insert to your recipient plasmid. We recommend...

...important plasmid features (such as the gene/insert, fusion proteins, point mutations, deletions, etc.) involves...

...backbone manufacturer's suggested leader sequence fused to your gene of interest, in frame with the start...

... before the cells begin dividing, the bacteria diffuse through the liquid and won't grow in colonies. ...

...settle to the bottom of the gel well, instead of diffusing in the buffer. Once solidified, place the agarose...

...Viral delivery methods include focused ultrasound (FUS), intracorporeal (IC), intracerebroventricular (ICV...widely throughout the organism to target large or diffuse biological structures. Of primary interest here...Stanton, A. C., Lagerborg, K. A., Tellez, L., Krunnfusz, A., King, E. M., Ye, S., Solomon, I. H., Tabebordbar...Stanton, A., King, E. M., Ye, S., Tellez, L., Krunnfusz, A., Tavakoli, S., Widrick, J. J., Messemer, K...

...nuclease specificity using truncated guide RNAs. 2014. Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. Nat Biotechnol...induced by CRISPR-Cas nucleases in human cells. 2013. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung...also be fused with transcriptional repressors or activators, and targeting these dCas9 fusion proteins...increase HDR frequency. Additionally, Cas9-CtIP , a fusion of Cas9 and CtIP, a protein involved in double-...editors (ABEs) . Cytosine base editors are created by fusing Cas9 nickase or catalytically inactive “dead” Cas9...cytosine base editors, the evolved TadA domain is fused to a Cas9 protein to create the adenine base editor...have been made by optimizing expression of the fusions, modifying the linker region between Cas variant...

...gene activation when fused to dCas9. In the dCas9-SunTag-VP64 system, dCas9 is fused to a scaffold containing...hook proteins fused to a streptavidin core that are able to retain reporter proteins fused to the streptavidin-binding...interest is fused to an sgRNA used to direct Cas9 to a specific genomic locus. When fused to the sgRNA...fluorescent protein fusions that can be used for monitoring cellular dynamics. These fusion proteins have been...When fused to an RNA of interest, Broccoli-DHFBI fluorescence can be used to visualize the fusion in vitro...or activation, the c-terminal cas9 fragment was fused with FK 506 binding protein 12 (Cas9(C)-FKBP-2xNLS...light-activated CRISPR-Cas9 effector, Polstein and Gersbach fused the light-inducible protein domains CibN and Cry2...

...single fusion protein, as in a FRET biosensor, then the local concentration of FPs around that fusion protein... can lead to polymerization of the fusion protein. A dimeric fusion protein will have two FP binding domains...polymer formation as fusion protein concentration increases. The polymerized fusion proteins that form ... blogger Erik L. Snapp. Stop using EGFP/GFP for fusion proteins! Despite multiple studies in high profile..., there's no problem. You're OK. However, if you fuse your protein of interest (POI) to GFP to study the...noncovalently dimerize. That means that your POI fused to GFP or another fluorescent protein (FP) could...tetramers (i.e. DsRed) [3]. This is a problem for fusion proteins. One of the major applications of FPs ...

...of interest(POI’s) are fused to TAs and DBDs (see Figure 2a) (4). These fusions are then co-expressed ....g. transforming both fusion plasmids, or mating two single haploid yeast fusion plasmid transformants...period, proteins were successfully expressed as fusions that retain their individual activities (1). Researchers...effectively (3). In Figure 1 for instance, the DBD of the fusion protein shown is still able to bind to the upstream...containing the target sequence of the DBD. The DBD-POI fusion (commonly referred to as the “bait”, Figure 2b)... two POI’s interact, this will bring the POI-TA fusion (commonly referred to as the “prey”) in close enough... reconstitutes functional luciferase only when fusions of the two halves of the luciferase protein interact...

... of two fusion proteins: 1) the genomic anchor - an inactive, dead Cas9 protein (dCas9) fused to CIB1;...CIB1 and it was fused to both the N- and C-termini of dCas9; and 2) CRY2FL-VP64, a fusion of full-length...can also be achieved using single-chain fusion proteins by fusing Cas9 to photoreceptors. One prime example...- the CRY2 photolyase homology region (CRY2PHR) fused to a transcriptional activator domain (VP64 or p65...p65). Upon expression in the cell, the dCas9-CIB1 fusion binds to the target DNA sequence as directed by...the guide RNA (gRNA), while the CRY2PHR-activator fusion floats freely, depicted in the figure below. Once...tested a variety of combinations to optimize both fusion proteins. The best performing combination was NLS-dCas9...

...tagged BioID2 for N-terminal fusions and Myc tagged BioID2 for C-terminal fusions. Kim, et al. Mol Biol Cell...molecular biologists to fuse fragments of DNA together in a desired order and express fusion proteins in a target...SpyCatcher fusion that can be anchored to amylose resin for solid-phase synthesis of larger fusions through...to generate mAID fusions to endogenous genes. Cell lines with the appropriate fusions could then be selected...problems by fusing an RBP to the catalytic domain of RNA editing enzyme, ADAR. This RBP-ADARcd fusion protein... to easily create protein expression constructs fused with epitopes or fluorophores, generate expression...CRISPR-Cas9-based tool for targeting CpG methylation by fusing catalytically inactive Cas9 (dCas9) with the catalytic...

...epigenome-engineering tools - zinc finger nucleases and TALENs fused to epigenetic modifiers enabled epigenetic modifications...Researchers showed that TALE-TET1 constructs, which fused a TALEN to the Tet1 demethylase catalytic domain...various promoters. Other researchers additionally fused a TAL effector to LSD1 histone demethylase to demethylate...targeting much easier! With the help of guide RNAs, fusions between Cas proteins and the epigenetic modifier...CRISPR applications direct catalytically dead dCas9 fused to a variety of epigenetic modifiers to specific...Transcriptional activation p300 acetyltransferase dCas9 fused to the catalytic domain of p300 acetyltransferase...has deposited pcDNA3.1-dCas9-MQ1(Q147L)-EGFP, a fusion of dCas9 to a small DNA methyltransferase from ...

...proteins can interfere with fused protein function Under some circumstances, the fused fluorescent proteins ...immunohistochemistry methods, these proteins are routinely fused to ‘tag’ or ‘label’ proteins to facilitate its detection...could affect the properties or trafficking of the fusion protein (Kimple et al., 2013). We found that the...initially used a construct in which the hM4Di was fused to mCherry. To investigate the localization of the...neurons, we next hypothesized that a different tag fused to the hM4Di might improve the transport of this...the influenza virus, introduced to the field of fusion proteins more than thirty years ago (Field et al...also found that this is not generalizable to all fusion proteins. For example, we saw that another type...

... NLuc fused to enhanced green fluorescent protein (GpNluc) and the other consists of NLuc fused to the...produce a series of FP-RLuc8 fusions for multicolor imaging. In all of these fusions coelenterazine intiates...plasmid #85203) NLuc fusions. The Nagai lab has additionally deposited more than 25 fusions of the Enhanced... Many of our technology platforms like HaloTag® Fusion Protein, codon-optimized Firefly luciferase genes...better biosensors. NanoLuc®-fluorescent protein fusions for imaging Fluorescent proteins are fantastic ...the more penetrative orange fluorescent protein fusion (OgNLuc). Both fluorescent proteins can be excited... by Nluc emission and the lab has dubbed these fusions LumiFluor Reporters. The LumiFluor reporter constructs...