...consistently throughout cells. VPR Description VPR fuses a tripartite complex with dCas9 to activate transcription... SunTag, SAM, and VPR have all shown significant improvements upon the initial dCas9-VP64 method, so there... cell lines.   dCas9-VP64 Description CRISPR activation can occur through fusing dCas9 with VP64, a strong...Generally, VPR is found to have significantly higher activation levels than the initial dCas9-VP64 activator... for CRISPR-based gene activation. dCas9-CBP In this method, dCas9 is fused to CBP, an histone acetyltransferase... the programmable guide RNA. Gene activation by dCas9, also referred to as CRISPRa, was initially published...methods for CRISPR activation CRISPR activation uses dCas9, a CRISPR protein variant lacking its endonuclease...

...(vector 1) with neuron-specific expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed...sgRNA (top) and neuron-specific expression of dCas9-VPR (bottom). Created with BioRender.com. The .... Co-transduction of primary rat neurons with dCas9-VPR and sgRNAs targeting either transcript variant..., 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...coding sequence. ES cells that stably express the dCas9-Suntag activator were transduced with a pooled sgRNA...Katherine E., et al. "A neuron-optimized CRISPR/dCas9 activation system for robust and specific gene regulation...

... 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...multiplexed gRNAs and of dCas9 fusions. The dCas-KRAB activator and the dCas-VPR repressor are introduced...approaches rely on continual expression of both dCas9 and the gRNA. This expression can vary significantly... could quantify and track cells expressing both dCas9 and gRNAs. The multi-gRNA piggyBac vector ...plasmids for piggyBac gRNA delivery and the all-in-one dCas9 targeting plasmids! Hazelbaker et al., Sci Reports...

...creating a new dCas9-VPR expression plasmid that uses the piggybac transposase to integrate dCas9-VPR into the...is genomic integration of dCas9-VPR and controlled expression of the dCas9-VPR transgene by doxycycline...the growing collection of dCas9-VPR plasmids, which already includes dSaCas9-VPR in an AAV transfer vector...activator developed by the Church lab, termed “dCas9-VPR”, which facilitates robust activation of target...than standard dCas9-VP64 activators) when introduced into mammalian cells. Although the VPR activation domain...activate target genes, the relatively large size of dCas9-VPR limits one’s ability to package it into lentivirus...transfection precluding the need for virus. Furthermore, dCAS9-VPR expression is under the control of the TRE promoter...

...fluorescent dCas9-KRAB and dCas9-VPR for quantifying and tracking cells that express both the dCas9 construct...Saccharomyces cerevisiae. The system consists of dCas9, the mammalian transcriptional repressor Mxi1, and...accessible and the complex enters the nucleus where dCas9-Mxi1 can repress gene expression. In the dark, the...Caulobacter cells for ctrA gene expression using various dCas9. Image adapted from Guzzo et al., 2020. Caulobacter...Caulobacter genes using a catalytically dead Cas9 (dCas9) derived from Streptococcus pasteurianus  or the...essential cell cycle regulator. The lab expressed both dCas9 and the ctrA sgRNA and saw that the levels of ctrA...minutes in xylose (which controls expression of dCas9). The lab demonstrated the system can also be used...

...propagation and keeping that dCas9 sequence in the pool. According to this logic, ω-dCas9 variants recognizing...With an NGG PAM, xCas9 3.7-VPR constructs modestly outperformed SpCas9-VPR in activating transcription... phage (SP) to carry catalytically dead SpCas9 (dCas9) fused to the bacterial polymerase subunit ω. This...NNN PAM sequences adjacent to a protospacer. If ω-dCas9 could recognize a library PAM, it would induce transcription...

...domains onto dCas9 to generate a dCas9-VP64-p65-Rta fusion protein (referred to as dCas9-VPR) had a synergistic...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...greater than the original dCas9-VP64 activator. They also revealed that dCas9-VPR mediated gene activation...proven challenging using the existing dCas9-VP64 activator. The dCas9-VPR activation plasmids have great potential...transactivating capabilities of CRISPR/dCas9-fusion proteins and have made their dCas9 transactivators available through... through Addgene. The development of dCas9 and dCas9-fusion proteins has dramatically increased the potential...

...including pcDNA-dCas9-p300 Core and pcDNA3.3-Nm-dCas9-p300 Core, as well as pLV-dCas9-p300-P2A-PuroR for...pCMV-dCas9-D3A) and Tet-dependent (TetO-dCas9-D3A) constructs. For lentiviral expression, Fuw-dCas9-Dnmt3a...Fusing dCas9 to a transcriptional activator like VP64 or VPR activates transcription, whereas dCas9-KRAB...Goodell's lab has deposited pcDNA3.1-dCas9-MQ1(Q147L)-EGFP, a fusion of dCas9 to a small DNA methyltransferase...activators/repressors - dCas9-p300 increased transcriptional activation more than dCas9-VP64, especially when...Non-editing 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...

...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...pX458 backbone ( Addgene #48138 ) dCas9 + KRAB-MeCP2 ( Addgene #110821 ) VPR ( Addgene #63798) Plasmids from...

... editor targets DNA (via catalytically inactive dCas9) to modify chromatin. A fluorescent protein can ...Jacobs-Wagner’s lab produce different levels of dCas9 expression and contain different antibiotic resistance...activation, repression, and editing with a single Cas9-VPR in Saccharomyces cerevisiae. To control Cas9 activity...

...environmental triggers. This is done by creating a split dCas9 protein that can reassemble when exposed to specific...Tanenbaum et al., 2014, Gilber et al, 2014), and VPR (Chavez et al., 2015)) are good choices across multiple...out our blog post covering VP64, Suntag, SAM, and VPR. Worry less about off targets Unlike Cas9 cutting...

...variants, such as a transcriptional activator dCas9-VPR, if these variants are fused to the MLV Gag in...

... applications, including CRISPR activation by dCas9-VPR, prime editing, and delivery of disease-relevant...

...imaging of proteins in living cells (Figure 9B) dCas9-VPR - dCas9 fused to several different activation domains...achieved by using orthogonal dCas9s (e.g. S. pyogenes dCas9 and S. aureus dCas9) tagged with different fluorescent...human cells. The simplest dCas9-based activators and repressors consist of dCas9 fused directly to a single...utilizes biotin tagging of dCas9 by fusing a biotin acceptor site to dCas9 and co-expressing BirA biotin... defense CRISPRa CRISPR Activation; using a dCas9 or dCas9-activator with a gRNA to increase transcription...target gene CRISPRi CRISPR Interference; using a dCas9 or dCas9-repressor with a gRNA to repress/decrease transcription...one strand. Catalytically inactive “dead” Cas9 (dCas9) contains mutations in both nuclease domains, D10A...

...imaging of proteins in living cells (Figure 9B) dCas9-VPR - dCas9 fused to several different activation domains...achieved by using orthogonal dCas9s (e.g. S. pyogenes dCas9 and S. aureus dCas9) tagged with different fluorescent...human cells. The simplest dCas9-based activators and repressors consist of dCas9 fused directly to a single...utilizes biotin tagging of dCas9 by fusing a biotin acceptor site to dCas9 and co-expressing BirA biotin... defense CRISPRa CRISPR Activation; using a dCas9 or dCas9-activator with a gRNA to increase transcription...target gene CRISPRi CRISPR Interference; using a dCas9 or dCas9-repressor with a gRNA to repress/decrease transcription...one strand. Catalytically inactive “dead” Cas9 (dCas9) contains mutations in both nuclease domains, D10A...