...reptiles at all. But new success is coming with CRISPR/Cas in lizards. The Menke Lab was able to make an albino...   It’s not an overstatement to say that CRISPR/Cas9 editing is one of the key tools now providing access... be immortal. Manihot esculenta (Blake Meyers) Cassava is the fifth most grown crop in the world so it...-Mediated Ovary Transduction of Cargo)  delivers Cas9 RNP to the arthropod germline by injection into ...

....2018.04.011 Polstein LR, Gersbach CA (2015) A light-inducible CRISPR-Cas9 system for control of endogenous...CRISPR/Cas9 is an enormously plastic tool and has taken the scientific world by storm. While Cas9 has been...required for Cas9-based activation. CRISPRa for genome-wide studies One of the best uses for Cas9 activators...context of the gene, Cas9 activators efficiently solve these problems.   Which Cas9 activator should I ...Best Cas9 Variant for Your Next Experiment Find out More about Genome-wide Screens with CRISPR/Cas9 Learn...with AAV Resources on Addgene.org Find Cas9 Activators Find Cas9 Repressors Catch Up on Your CRISPR Background...there has also been significant work on constructing Cas9 transcriptional activators. These constructs allow...

...its high resistance to antibiotics. Several CRISPR/Cas-based systems have been developed for other mycobacteria...Overview of the dual-plasmid CRISPR/Cas9 workflow in M. abscessus. A) Sth1Cas9 mediates double strand break ...mScarlet-based tags for live imaging in C. elegans CRISPR/Cas9 for gene disruption in M. abscessus  Bioluminescent...Mycobacterium abscessus using a dual-plasmid CRISPR/Cas9 system By Aryana Khosravani Deborah Hung’s lab has... has developed a novel dual-plasmid CRISPR/Cas9 system for gene disruption in Mycobacterium abscessus,...resistance interfering with selection. Using an inducible Cas9 expression and removing the sgRNA plasmid after ...Figure 4). This work marks the first use of CRISPR/Cas9 in M. abscessus. The simplicity and efficiency of...

...RNA-guided editing of bacterial genomes using CRISPR-Cas systems." Nature biotechnology31.3 (2013): 233. PubMed...expression of a gene with an antibiotic resistance cassette. This leaves a “scar” in the chromosome, sometimes...(Thomason et al., 2014). In recent years, CRISPR-Cas9 technology has also aided in the creation of scarless...vector contains sacB, an chloramphenicol resistance cassette, and the R6K origin of replication to allow replication...amount of user-to-user variability, and in some cases, counter-selection escape can occur: single crossover...pDS132 (Philippe et al., 2004): Replaced the sacB cassette with one of three toxins from the Khetrapal paper...15109831. Pyne, Michael E., et al. "Coupling the CRISPR/Cas9 system with lambda red recombineering enables simplified...

...revolution in genome editing methods (most notably CRISPR/Cas mediated HDR) enables the tagging of endogenous genes...are necessary for its native properties. In these cases, an insertion of the fluorescent protein into the...

...editing in rice. In Genome Engineering: The CRISPR-Cas Revolution, Cold Spring Harbor Laboratory (2018)....by tethering cytosine deaminase with Cas9 nickase (nCas9). nCas9 creates a nick in the DNA by cutting ...base editors replacing the SpCas9 with the mutant versions of SpCas9 and SaCas9, which recognize PAM sequences...Nickase Cas9 (D10A) Deaminase PAM requirements Reference CBE SpCas9 rAPOBEC1 NGG 6 SpCas9 Target-AID...PmCDA1) NGG 5 SpCas9-VQR rAPOBEC1 NGA 8 SaCas9-KKH rAPOBEC1 NNNRRT 8 ABE SpCas9 TadA-TadA* NGG 9... 9-12 SpCas9-VQR TadA-TadA* NGA 8 SaCas9 TadA-TadA* NNGRRT 9 SaCas9-KKH TadA-TadA* NNNRRT 8 SpCas9...tRNA adenosine deaminases (ecTadA), fused it with dCas9, and transformed it into E. coli harboring the defective...

...https://doi.org/10.1155/2012/980250  Bates A, Power CA (2019) David vs. Goliath: The Structure, Function...

...Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. Enzymatic assembly of DNA molecules...modularity to overlap-directed methods (Casini 2014). In this case linker sequences are added before the...In this case, rather than using a restriction enzyme or PCR to make linearized vector, the Cas9 enzyme ...standard sequences with overlap regions, such as in the case of MODAL (modular overlap-directed assembly with...restriction site for linearization. This is a perfect case for the use of Gibson assembly combined with the...assembled into a vector using Gibson assembly. In this case the vector was PCR amplified to contain regions ...Li K, Wang B, Jin S, Reiser M, Lockey RF. CRISPR/Cas9 nuclease cleavage combined with Gibson assembly ...

...loci in living human cells by an optimized CRISPR/Cas system." Cell 155.7 (2013): 1479-1491. PubMed PMID...anaerobic conditions needs to be examined on a case-by-case basis. GFP is sensitive to acid The chromophore...nuclease-deficient Cas9 can bind loci in the genome that are defined by a gRNA. Since Cas9 is a protein, a...the localization of the fusion protein. Also, in cases where the protein of interest is relatively small...the function of the protein. This is clearly the case when proteins undergo post-translational modification...

...High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology, 31...using CRISPR/Cas9 to edit a gene, the expression of Cas9 is no longer necessary. However, Cas9 can be toxic...like creating stable cell lines, delivering CRISPR/Cas9 to cells for genome editing, and introducing inducible...t uncommon. Many naturally occurring viruses occasionally integrate their genomes into those of the cells...

...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...

...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...

...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...