We narrowed to 48 results for: dcas9 coding sequence

...Cas9 known as dCas9 (Jinek et al., 2012). Like Cas9, dCas9 can bind to a specific DNA sequence via a targeting...targeting gRNA. But dCas9 does not cleave the DNA. Much of the research using dCas9 has focused on transcriptional...mammalian cells by expressing dCas9 with an N-terminal 3xFLAG tag (3xFLAG-dCas9) and a gRNA targeting their...through a specific gRNA-dCas9 complex. When combined with next-generation sequencing, this method allows ... of non-coding RNAs associated with telomeres using a combination of enChIP and RNA sequencing." PLoS ...activators (Qi et al., 2013). However, the fusion of dCas9 with a protein tag allows for the isolation of a...genomic region using the CRISPR system, consisting of dCas9 and targeting gRNA, to identify molecular interactions...

...non-mammalian sequence (GAL4) was supplied. In addition to labeling repetitive telomeric sequences, Chen et...ortholog requires a different PAM sequence, a gRNA designed for one dCas9 should be specific to that ortholog... et al. tested their dCas9 variants using gRNAs specific for telomeric sequences and showed that different...fluorescently labeled dCas9s are efficiently directed to the proper target sequence. They succeeded in labeling...specified by PAM sequence, each color requires a different Cas9 ortholog, and the target sequence must be located...binding domain sequence (as noted in the table). gRNAs with two different hairpin sequences produce yellow...bright-field image. Image from Ma et al, 2016. Using dCas9 to visualize genomic loci in vivo The flexibility...

...with dCas9, this new scaffold RNA (scRNA) will bind to a PAM-adjacent target nucleotide sequence then .... Using dCas9 as an on/off switch The multiple scRNAs all individually utilize the same dCas9 protein....modulate dCas9 expression, multiple scRNA transcriptional programs can be regulated with dCas9 as a master...an orthogonal dCas9 protein that will still recognize guide RNA but with a different dCas9 binding module... consists of a catalytically dead Cas9 protein (dCas9) that lacks endonuclease activity coupled with a...sgRNA) that bears complementarity to a 20bp target sequence (Qi et al. 2013). Modifying the CRISPRi system...al. 2015).  These scaffolds are stretches of non-coding RNA interspaced with linkers that can fold into...

...genes using dCas9-based activators The simplest of the dCas9 activators consists of dCas9 fused to a single...site early in the coding region of the gene of interest. This gives you loads of sequences to select from...Repressing target genes using dCas9-based repressors Early experiments using dCas9 in bacteria demonstrated... Fig. 3: Cas9 repressors include dCas9 alone and a dCas9-KRAB fusion, both targeted to the promoter...sgRNA), provided it meets two conditions:  1) The sequence is unique compared to the rest of the genome.  ...upstream of a Protospacer Adjacent Motif (PAM), the sequence NGG.  If these conditions apply to your experimental...feasible for your experiment, ensure your sgRNA sequence has little homology to other regions of the genome...

...downstream of the TUJ1 coding sequence. ES cells that stably express the dCas9-Suntag activator were transduced..., CRISPRa uses a nuclease-dead variant of Cas9 (dCas9) and engineered combinations of transcriptional ... transcriptional activator to each genome-bound dCas9 (see Figure 1a for the transgenic construct). More...mCherry (vector 1) with neuron-specific expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed...of sgRNA (top) and neuron-specific expression of dCas9-VPR (bottom). Created with BioRender.com. The...the same gene but with different untranslated or coding regions. Typically, each gene isoform has a distinct...investigation. Co-transduction of primary rat neurons with dCas9-VPR and sgRNAs targeting either transcript variant...

...specific genes using dCas9-VP64 activator fragments (dCas9(C)-FKBP-2xNLS-VP64 and dCas9(N)-FRB-NES). This...gene activation when fused to dCas9. In the dCas9-SunTag-VP64 system, dCas9 is fused to a scaffold containing...domains onto dCas9 to generate a dCas9-VP64-p65-Rta fusion protein (referred to as dCas9-VPR) had a synergistic...co-expressed with dCas9 – the dCas9, sgRNA/RNA cargo complex localizes to the sequence targeted by the ...aptamer, and more - to sequences on a Gaussia luciferase reporter vector. dCas9 or dCas9-VP64 (a control that...initiation sequence known as the Species-Independent Translational Sequence (SITS). This sequence bypasses... via DNA sequencing or other readout. Cellular memory is accomplished by the use of sequence-specific ...

...an inactive RNA-guided DNA binding protein (dCas9). dCas9 is then tethered to a repressive domain, such...loss of function while nuclease-deactivated Cas9 (dCas9) can be tethered to a repressor (CRISPRi) or an ...examined approximately 1,900 sgRNAs and identified sequence features and adjustments that improved sgRNA activity...with an average of 4 sgRNAs per gene and 1,000 non-coding control sgRNAs. This library was designed to improve...library In addition to inhibiting gene expression, dCas9 can also be used for transcriptional activation....developed to recruit transcriptional machinery to dCas9 and its RNA directed binding site. These methods... fusion of an activation domain such as VP16 to dCas9 or the recruitment of multiple copies of VP16 termed...

...effective targeting either the promoter or the coding sequence and the template or non-template strand, among...targeting either a catalytically dead Cas9 (dCas9) alone or a dCas9 fused to the omega subunit of RNA polymerase...tuberculosis. dCas9 and a gRNA are expressed on pRH2502 and pRH2521, respectively. Both dCas9 and the gRNA...fused to dCas9 and efficiently introduce cytosine-to-thymine substitutions in target sequences (Banno et...recombineering is unsuccessful and the wild-type sequence persists, Cas9 will cleave the gene of interest...have inactivating mutations in the Cas9 or spacer sequence, and these rare events are easily detectable using...2013). As in other systems, catalytically dead (dCas9) targeted to a promoter or gene body can repress...

...target the coding or non-coding strand of DNA. As a rule of thumb, we avoid target sites that code for amino...can worry less about sequence optimality because you generally have fewer sequences to choose from). Finally...location and sequence are of approximately equal importance in design – an optimized sequence will do little...modification. Whole-genome sequencing of cells modified by CRISPR indicates that the consequences of off-target activity...or modulation of gene expression. Location and sequence are important considerations for designing your...you target, but it is important that your gRNA sequence is designed to be highly active and reduce off...means there are so few gRNAs to choose from that sequence preferences must largely be ignored. Created...

...self-transcribing active regulatory region sequencing) that tests enhancer sequences outside of their endogenous chromatin...containing many different promoters, RBS’, coding sequences (CDS), and terminators of varying strengths..., and these sequence changes are subsequently identified through transcriptome sequencing. Importantly...creating a new dCas9-VPR expression plasmid that uses the piggybac transposase to integrate dCas9-VPR into ... is genomic integration of dCas9-VPR and controlled expression of the dCas9-VPR transgene by doxycycline...generated 5 common 30bp overlap sequences (Sites #1-5). Each overlap site encodes a unique restriction enzyme... due to the variable availability of enhancer sequences in different chromatin states. Alexander Stark...

...inactive dCas9 and microbial SSAPs (dCas9-SSAP) which insert genes in a cleavage free manner. The dCas9-SSAP... (M-CREATE) dCas9-SSAP with reduced error rate for long sequence knock-ins Visual barcodes for live cell...10.1038/s41593-021-00969-4.   dCas9-SSAP with reduced error rate for long sequence knock-ins by: Lucie Wilson...lab has developed a method using dCas9-SSAP to knock-in long sequences with reduced risks of unwanted mutations...into an AAV system. dCas9-SSAP will make the editing of kilobase-scaled sequences more efficient and less...cleavage-free dCas9-based gene editor using microbial SSAPs. Image from Wang, et al, 2022.  Find dCas9-SSAP... mutations in mammalian cells. Long sequences traditionally have higher risks of off-target mutations....

... plasmids dCas9-KRAB-MeCP2 fusion for gene and non-coding enhancer sequence repression dCas9-VPR fusion...175574 express dCas9-KRAB-MeCP2 for repression. Plasmids 175571 and 175572 express dCas9-VPR for activation... Plasmid and Sequence Derivative Sources Original pX458 backbone ( Addgene #48138 ) dCas9 + KRAB-MeCP2...activation. Usage Guide sequences can be designed using Benchling or using similar design tools and ordered...Golden Gate cloning to insert the desired guide RNA sequence by digestion of the backbone with BbsI followed...phosphorylated oligonucleotides containing the guide target sequence: Up to two guides can be expressed from the same...

...roll please… non-coding RNAs! You probably didn’t see that coming, right? Non-coding RNAs (ncRNAs) are...disable, protein-coding genes by generating insertions or deletions in the DNA sequence. This same approach...High-Throughput Characterization of Long Non-Coding RNAs. Non-Coding RNA. 2021;7(4):79. doi:10.3390/ncrna7040079...heard that only 2% of our genome is made of protein-coding genes, and you might be wondering what the rest...functionally manipulate and study in comparison to protein-coding genes (Rinn and Chang 2012). Scientists have determined... determined the function of many protein-coding genes thanks to systems like CRISPR/Cas9 that allow them...very small deletions and insertions in the DNA sequence can completely disrupt expression of the transcribed...

...guide RNA (gRNA) sequences. But these approaches rely on continual expression of both dCas9 and the gRNA....an EGFP reporter. Expression of the dCas9-KRAB activator or dCas9-VPR repressor in hPSCs is also driven...piggyBac gRNA delivery and the all-in-one dCas9-KRAB and dCas9-VPR targeting plasmids can be found at Addgene...transfected with plasmids encoding FPs tagged with the plasma membrane targetin sequence derived either from...evolution techniques mimic natural selection of sequences that evolve a measurable property of a protein...of tobacco etch virus protease (TEV), a highly sequence-specific protease used to cleave affinity tags...plasma membrane, they tested different target sequences for improved selectivity and found that the best...

... protein dCas9 is expressed along a CRISPR array that contains 20 bp "addresses", where dCas9 is going...going to bind on the chromosome. When dCas9 binds to a gene, it blocks the progression of the RNA polymerase...Mismatches between the guide RNA and the target sequence lower the repression level. By controlling the...for quick integration of the aTc-inducible pTet-dCas9 in the attachment site of the phage 186. Detailed... Bikard, D., & Van Teeffelen, S. (2018). Tuning dCas9’s ability to block transcription enables robust,...Schematic of the strain expressing two reporters and dCas9 under a P-tet promoter integrated in the chromosome...be controlled using a plasmid‐borne CRISPR array coding for guide RNAs (diamonds) interspaced with CRISPR...

...dead Cas9 (dCas9) based activators (such as dCas9-VP64) or repressors (dCas9-KRAB or dCas9-SRDX) can also... a ~20 nucleotide targeting sequence and ~75 nucleotide scaffold sequence) is consistent between plants...consider using genome editing to change the genetic code of plants, including the development of crops with...contain a plant pol III promoter and gRNA scaffold sequence and allow researchers to insert targeting oligos...is commonly tagged with a nuclear localization sequence to enhance targeting to the nucleus, and several...the specifics - promoters used, precise protein sequences or domains, and methods of delivery - the underlying...

...expression in the cell, the dCas9-CIB1 fusion binds to the target DNA sequence as directed by the guide ...in which dCas9 binding occurs. Bubeck et al. expressed a telomere-targeting gRNA and a dCas9 fusion protein.... sphaeroides Cas9, dCas9 Gene editing Zhou et al., 2018   pdDronpa Cas9, dCas9 Gene editing, transcription...Shining light on transcriptional activation using dCas9 Initial photoactivatable CRISPR systems published...genomic anchor - an inactive, dead Cas9 protein (dCas9) fused to CIB1; and 2) the activator - the CRY2 ...proteins. The best performing combination was NLS-dCas9-trCIB1 and NLSx3-CRYPHR-p65 - it had the lowest ...The optimized LACE system consisted of: 1) CIBN-dCas9-CIBN, where CIBN is the N-terminal fragment of CIB1...

...genome sequence dCas9 or dCas9 fusion with transcriptional repressor (such as dCas9-KRAB) dCas9-KRAB can...modifying the genome sequence dCas9 fusion with transcriptional activator (such as dCas9-VP64) Many different...perfect world, your gRNA sequence would be an exact complement to your target sequence with no homologous sites... the desired target sequence, or on-target activity. Two gRNA targeting sequences with 100% homology to... selected target sequence. Additionally, any mismatches between a given gRNA sequence and your genomic...customization Usually paired with a sequencing method Can’t provide specific sequence information alone Digital ...rare edits Many options like RNA-sequencing, Perturb-seq, deep sequencing, etc. qPCR Knockouts Activation...

...options include humanized wt Cas9, dCas9 (transcriptional repressor), and dCas9-VP64 (transcriptional activator...D10A nickase mutant (Cas9n), dCas9 (transcriptional repression), and Fok1-dCas9 (dimeric nuclease). Gateway...Deleting a large region of the genome by removing the sequence between two target sites. Modifying multiple genes... oligonucleotides for your chosen gRNA target sequences and clone them in just as you would for a single...enzymes, which cleave outside of their recognition sequence, creating flanking overhangs. These overhangs ...the oligonucleotides specifying each gRNA target sequence into distinct expression vectors using the enzyme...the promoter-gRNA construct, but with different sequences adjacent to the sites. When digested with the ...

...Plant Yeast Resources Catalytically dead Cas9 (dCas9), or dCas9 fused to a transcriptional repressor peptide...promoter, enhancer, or the beginning of the coding sequence. Figure 1: Overview of CRISPR interference....