...carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering." Cell 154 (2013):1370... 47327 pET-28b-Cas9-His 6xHis (C terminal); NLS (C terminal) 62933 pET-Cas9-NLS-6xHis 6xHis.... recommend ribonucleoprotein (RNP) complexes of Cas9 protein and the separated guide RNA components (..., we’ll use the example of inserting a 2A-FlpO cassette into the Fgf8 gene. Miura et al. (2018) suggest...insertion site to maximize HDR efficiency. In this case, we’re fortunate that there’s a guide that directs... and cleavage sites (slashes) are shown. In this case, the green guide would be selected, since it cleaves...increase T7 promoter activity. To avoid re-cutting by Cas9 after editing, make sure that the repair template...

...Biotechnology.       References Degeling MH, Maguire CA, Bovenberg MSS, Tannous BA (2012) Sensitive Assay...to discard the infected cell culture flasks. In cases where the culture is irreplaceable or very expensive...

...in Vibrio cholerae that encodes a Type I-F CRISPR-Cas system. This system has four major components: 1)...crRNA), 2) four proteins (TniQ, Cas8, Cas7, Cas6) that forms the QCascade DNA-targeting module with the ... transposons encode reporter genes, mutagenic cassettes, or barcodes. When transposons are delivered to...For the INTEGRATE system to transpose, first the QCascade complex surveys the genome and binds its target...molecule, and inserts it ~50 bp downstream of QCascade’s genomic target site (Fig. 3B). The INTEGRATE ...of RNA-guided transposition with INTEGRATE. The QCascade complex surveys the genome and binds its target...molecule, and inserts it ~50 bp downstream of QCascade’s genomic target site.  T-RL= transposon right-...

...allow positively charged ions such as Na+, K+, and Ca++ to passively enter the cell with the concentration...function through multiple intra-cellular signaling cascades. Optical methods for controlling GPCR activation...control GPCR activation and downstream signaling cascades (reviewed in Abreu & Levitz, 2020), including ...extracellular domain which activates the native signaling cascade of the intracellular part of the receptor, similar... out important branch points along a signaling cascade that may go awry in a disease state.      ...

...Find out more at www.projbridge.com. Walnut Creek, CA, USA 37.9101°N 122.0652°W – Steph Hays When I marched...22 to join in the March for Science.  Cold and overcast with intermittent rains, it was weather befitting...

...Addgene. 10. Port, Fillip, et al. “Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering...humanized Cas9, D10A nickase mutant (Cas9n), dCas9 (transcriptional repression), and Fok1-dCas9 (dimeric...options include humanized wt Cas9, dCas9 (transcriptional repressor), and dCas9-VP64 (transcriptional activator...including Standard PolIII-gRNA cassette, Csy4-cleavable cassette, and PTG cassette.    Find gRNA multiplexing... then digested using BsmBI and ligated into Cas9 or dCas9-containing destination vectors. Destination ... encoding the gRNAs and Cas9. As with the Gersbach lab plasmids, multiple Cas9 variants are available:...the multiple gRNAs and Cas9 together into a single plasmid. Although only wt hCas9 entry vectors are supplied...

...either Neo or Hygro resistance markers with CRISPR/Cas vectors targeting their genes of interest to generate...CRISPR-Cas9-based tool for targeting CpG methylation by fusing catalytically inactive Cas9 (dCas9) with...pDGE Dicot Genome Editing Kit contains Cas9, Cas9 nickase, and Cas9 activator expressing plasmids that can...high-throughput CRISPR/Cas9 gene modification in Caenorhabditis elegans CRISPR/Cas9 technology has revolutionized...reported on a novel Cas9-based activator developed by the Church lab, termed “dCas9-VPR”, which facilitates...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...

... potential applications. Polstein LR & Gersbach CA, Nat Chem Biol 2015 Mar;11(3):198-200. SunTag system...fully functional Cas9 upon dimerization. Splitting wild-type Cas9 into n-terminal (Cas9(N)-2xNLS) and c-terminal...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... can write a blog post about it! Split Cas9 system The Cas9 protein is composed of an n-terminal DNA... the bi-lobed architecture of Cas9 to engineer a series of "split" cas9 molecules that cannot function...

... gRNA, Cas9 binds the target site, but does not cut either strand of DNA. Traditional Cas9 regulators...capability, and this nuclease-null or “dead” Cas9 (dCas9) variant can then be fused to effector domains...importantly, most Cas9 orthologs are less well characterized than the conventionally used SpCas9 (derived from...to exhibit more limited activity than SpCas9 (2). In many cases this is due both to lower relative nuclease... a potent Cas9-based transcriptional regulator without making any functional changes to Cas9. For instance...repression with nuclease-competent Cas9. When complexed with a truncated gRNA, Cas9 is still able to bind DNA ...on rare occasions truncated gRNAs (≤15nt) have been found to retain some ability to induce Cas9 to make...

... can occur due to prolonged Cas9 expression     The requirement for Cas9 transcription and translation...consisting of the Cas9 protein in complex with a targeting gRNA, to your cells of interest. Cas9 RNPs are capable...pathways. This makes Cas9 RNPs useful for CRISPR applications where limited expression of Cas9 is required and...long-term expression of Cas9, such as visualizing genomic loci using fluorophore tagged dCas9 may require the ...express and purify His-tagged Cas9 from E. coli using a plasmid such as pET-28b-Cas9-His from Alex Schier’s ... of Cas9-gRNA RNPs by specific cell types (Rouet et al. 2018). This was accomplished using a Cas9 mutant...for experiments where stable Cas9 expression is required, such as when a Cas9 fusion to a fluorescent protein...