...researchers injected Cre-expressing AAV-5 into the DRN of a transgenic mouse harbouring a Cre-dependent IL-1...injected AAV2 particles into a Drd1-Cre transgenic mouse to deliver Cre-dependent expression of  Channelrhodopsin...-1 beta overexpression cassette. Expression of Cre in these neurons turns on overexpression of IL-1 beta... (Howerton at al. 2014). Addgene has several recombinase-expressing ready-made AAV vectors which can be...2014). Scientists from the University of Geneva (Creed et al., 2015) used optogenetics to support these...transmission from the mPFC projections to the NAc and decreased the locomotion of the animal. This confirms the...behavior in rats in the forced swim test as seen by increasing the ratio of time spent actively swimming versus...

... at Addgene New serotypes for the Cre-dependent Flpo recombinase pEF1a-DIO-FLPo-WPRE-hGHpA, which is...sensors include syn-jYCaMP1s (slow variant), the Cre-dependent syn-FLEX-jYCaMP1s, and the axon-targeted...syn-FLEX-axon-jYCaMP1s. Find these AAVs at Addgene Cre-dependent EYFP AAV in the new serotype PHP.V1 that...biotechnology tool to optimize another. The authors have created a yeast-based directed evolution approach with ...your studies.  Find the plasmids to set up your screening systems at Addgene! Schematic of the evolution...following a DNA miniprep protocol!  First, the authors created the pMAK backbone by modifying pMAL-T-Avi-His/BirA...exported to the periplasm of bacteria where it can be secreted into the supernatant. Four plasmids containing...

... The old standby of CRE/flp didn’t work – it turns out these tyrosine recombinases are as bad at integrating...likely do it using Prime editing alongside some creative (and significant!) protein engineering. They combined...insert being “dropped” in the wrong place, while increasing the number of potential applications. But they...

...AAV serotypes, and the ever-growing variety of recombinase systems. Picking Ligands Picking the right ligand...further modified to create ultrapotent PSEMS (uPSEMs) that have drastically increased potency. Download ...signaling pathways and can lead to such responses as decreased heart rate (Redfern et al., 1999) or altered neuron...firing. Other DREADDs, such as the KORD receptor, increase this threshold and silence neurons. DREADD-activated...actuator modules, were developed with the goal of creating receptors with less endogenous activation and ... Higher potency means lower drug doses, which decreases the risk of off target effects. These synthetic...

...dynamics in fine structures. We have expanded our recombinase-dependent virus selection to now include Flp-... aaRSs in these processes. To create the antibody toolbox, they screened antibody candidates using a synthetic...fourteen aminoacyl tRNA synthetases (aaRS) have been created, characterized, and validated by the Gräslund lab...cross-reactivity, antibody candidates underwent affinity screening to select for slow off-rate binders. The best ...pathways By Gabrielle Clouse Claudia Vickers’ lab has created a novel nanocompartment for transporting proteins...

...Plasmids: CRISPR transposases CRISPR Recombinases and Integrases Recombinases, and their subfamily integrases...for large scale edits. These proteins, like Cre recombinase or phage derived serine integrases, insert ...mechanisms, including transposons, integrases, and recombinases, with Cas enzymes. CRISPR Transposases Transposon...lab , pairs prime editing with a large serine recombinase (LSR). A prime editor is used to install the ...evoBxb1 or eeBxb1 are evolved versions of the recombinase with improved integration efficiencies in mammalian...& Hsu, P. D. (2022). Systematic discovery of recombinases for efficient integration of large DNA sequences... GAT; increase nuclease fidelity SpCas9-NG - NG; increase in vitro activity SpG - NGN; increase nuclease...

...vector assembly, and the remaining 5 plasmids include Cre and FLP expression vectors, a general cloning plasmid...selection markers are available. An improved FLP recombinase expression vector allows for efficient marker..._CEBPA_1 PX458_CEBPA_2 CREB1 Human FLAG pFETCh_CREB1 PX458_CREB_1 PX458_CREB_2 TGIF2 Human FLAG pFETCh_TGIF2...Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors gRNAs...genome modification. This system can be used to create C- and N-terminal epitope tags. The plasmids in...has developed a systematic and scalable method to create marker-free mutations, insertions, and deletions...stem cell (hiPSC) lines. These cell lines were created using CRISPR and the donor plasmids containing ...

...sequence of interest (red) is amplified using Recombinase Polymerase Amplification (RPA) or Reverse-transcription...dLwaCas13a as a programmable RNA-binding protein, creating the fluorescent fusion protein dLwaCas13a-NF for...

...resistance markers, and other enzymes such as Cre-recombinase drastically expanding the base-editing reporter...Base editors create specific point mutations in the genome, but they’re inefficient compared to CRISPR...experiments. In the past few years, scientists have created an array of base editing reporters that can help...Cas9-mediated editing in real time The Harris lab created ACE, a reporter that monitors APOBEC (a family ...and a downstream constitutively active eGFP. To create the inactive mCherry, the lab introduced a 43 base-pair...DSB in their previous reporter, the Harris Lab created a panel of eGFP reporters to quantify the on-target...complexes in real-time (St. Martin et al., 2019). To create the eGFP reporters the Harris lab individually ...

... (pharyngeal co-injection marker); pDD104 (Cre recombinase) PDF 102.5 KB Jaenisch CRISPR plasmid summary.... Green Listed - a CRISPR Screen Tool Design gRNAs for custom CRISPR screens targeting a long or short...labs who have deposited CRISPR plasmids , we've created a series of links and guides to help you use CRISPR...considerations, and more. CasPEDIA is a community resource created and maintained by the Doudna Lab at the Innovative...elegans, mouse, human, rat, cow, dog, pig, Thale cress, rice (Oryza sativa), tomato, corn, monkey (macaca...additional HDR & gRNA plasmids are available for CREB1, ATF1, GABPA, & RAD21 PDF 134.7 KB Musunuru CRISPRs...

...PMCID: PMC4165888. 6.Witten, Ilana B., et al. "Recombinase-driver rat lines: tools, techniques, and optogenetic...using Chr2 stimulation should result in robust increases in c-fos expression in the targeted brain region...

.... Flipbow employs the Flp recombinase/FRT system in place of Brainbow’s Cre/loxP, allowing for simultaneous...all-in-one versions which express self-excising Cre recombinase from the same transgene as the Brainbow cassette... short hairpin) and CRUSH (Conditional RUSH) use Cre-mediated recombination to turn on or off the expression...a dual-color RNAi “sensor” mouse strain in which Cre expression causes the induction of both DsRed fluorescence...Mice carrying the R26DsRedR; CRUSH-GFP; and Nestin-Cre alleles showed efficient GFP knockdown in eyes and... recombination events at the genetic level. Upon Cre/loxP recombination, each transgene expresses one ...SUMO tags in the FP sequence for separation from Cre-based Brainbow-expressing cells. Autobow plasmids...

... from a sample is first amplified using RPA (recombinase polymerase amplification) or reverse transcriptase...the presence of your targeted nucleic acid. To increase the sensitivity of the assay, targeted DNA or ...non-specific (trans) ssDNA and harnessed this ability to create a DNA detection platform called DETECTR. How does...

... the expression of a Cre recombinase contained within the knock-in. Including Cre-mediated marker excision...kilobases of additional sequence being introduced. Cre-mediated excision of the selective cassette minimizes...experimental manipulation. In some cases, injection of Cre is required, however the development of the self-...variable amounts of experimental manipulation and screening.  A co-selection strategy for CRISPR editing in... with inefficient sgRNAs in my initial direct screening efforts, which were similar to the approach detailed...low efficiency (0.13%) and laborious handling (screening 768 F1 animals). Recovering rare editing events...conditional-lethal mutation to identify edits with minimal screening effort. Selecting for repair of a lethal mutation...

...Plasmids: CRISPR transposases CRISPR Recombinases and Integrases Recombinases, and their subfamily integrases...for large scale edits. These proteins, like Cre recombinase or phage derived serine integrases, insert ...mechanisms, including transposons, integrases, and recombinases, with Cas enzymes. CRISPR Transposases Transposon...lab , pairs prime editing with a large serine recombinase (LSR). A prime editor is used to install the ...evoBxb1 or eeBxb1 are evolved versions of the recombinase with improved integration efficiencies in mammalian...& Hsu, P. D. (2022). Systematic discovery of recombinases for efficient integration of large DNA sequences... GAT; increase nuclease fidelity SpCas9-NG - NG; increase in vitro activity SpG - NGN; increase nuclease...

...and/or off-target effects (e.g. recombinase-dependent vectors) increases with the amount of virus you add...from Aschauer et al., 2013, Figure 1. Used under Creative Commons Attribution License. Within the nervous...can become methylated over time, resulting in decreased expression (Damdindorj et al., 2014; Nieuwenhuis...from Griffin et al., 2019, Figure 4. Used under Creative Commons Attribution 4.0 International License....vice versa. The total volume needed, however, increases with the size of the region you want to cover ...

...powerful when combined with site specific recombinase technology like Cre-lox, which allows you to express an...when one half of a given protein, such as the Cre recombinase, is fused to Cry2 and the other half to CIB1...using a viral vector with a floxed opsin in a VGAT-cre animal will result in expression of the opsin only.... Novel recombinant adeno-associated viruses for Cre activated and inactivated transgene expression in... include: Increased photocurrent amplitude Examples: ChR2(H134R), C1V1(t/t), ChIEF Increased channel (... following enhancements: Increased light sensitivity Example: ArchT Increased photocurrent amplitude Examples...Scherffelia dubia ). Synthetic variants have been created via genetic point mutations, codon optimization...

...lentiviral vector, but transient expression of Cre recombinase allows for deletion of the inserted transgenes...formation in iPSC-derived and iPSC-transplanted mice. Cre-deletable or Tet-inducible lentiviruses address some...considered safe for clinical applications. Excisable Cre-lox lentiviral vectors: This approach is the same...insertional mutagenesis. Random integration also creates heterogeneous iPSC cell lines, which can complicate...insertional mutagenesis and random integration creates variation between iPSC clones. Retroviruses are...which is removed by culturing cells at 38℃, but screening cells for the presence of transgenes is still ...

...specialized system with parts including site-specific recombinases, regulatory elements, developmental regulators... technology has been used to optimize protein secretion and expression in the biotech workhorse yeast ...Engineering disease resistance through gene stacking, increasing production of a protein of interest, and improving...genetic circuits with multiplexed guides, mini screening libraries, direct edit optimization, and more....can be invaluable for more complex editing and screening experiments in organisms that don’t yet have these...modify it as necessary. And remember, if you end up creating a really cool MoClo kit, we would love for you...Modular Toolkit for Generating Pichia Pastoris Secretion Libraries.” ACS Synthetic Biology, vol. 6, no....

...loxP-stop-loxP cassette into the Rosa26 locus, allowing for Cre-dependent expression of a GMAP inserted gene. The...species. Indeed, in order to use MAGE, the λ Red recombinase enzymes need to be expressed and the native methyl-directed...plasmids (dubbed pORTMAGE) expresses the λ Red recombinase enzymes, as well as a dominant-negative mutator...of a good screening experiment can be found on our blog post entitled “Genome-wide Screening Using CRISPR...Receptor-ome Expression and Screening via Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC-1 library...and later create stable cell lines expressing it. The Church lab fixed this issue by creating a new dCas9...  CRISPR pooled libraries enable researchers to screen the entire genome for genes that regulate a wide...