...efficiency. Finally, as shown in Drosophila S2 cells (Böttcher et al., 2014), inactivation of non-homologous...Originally published Jan 27, 2015 and last updated Dec 17, 2020. This post was contributed by Jordan ...heat-shocking to induce the expression of a Cre recombinase contained within the knock-in. Including Cre-...approach – co-CRISPR from Craig Mello’s lab – used inactivation of the unc-22 gene as their selection marker...Andy Fire’s lab used oligo-mediated knock-in of a dominant mutation, known as co-conversion (Arribere et ...by channeling DNA breaks into the homologous recombination repair pathway. Reagents required to perform...), and Victor Ambros and Gary Ruvkun for their seminal work on micro RNAs in C. elegans. The C. elegans...

...Lactose permease inactivated, lactose cannot be taken up by cell mcrA, mcrBC Inactivation of pathway that... is essential for recombination and general DNA repair Reduces plasmid recombination, increases plasmid...stability, reduced recombination relA or relA1 Relaxed phenotype, mutation eliminating stringent factor...them in 1885. E. coli are mainly found in the intestinal tract of animals. There are many different naturally... foreign (methylated) DNA mrr, Δ(mcrC-mrr) Inactivation of pathway that cleaves methylated adenine or...ara disruption of arabinose metabolism pathway Inability to utilize arabinose as a carbon source leuB...leuB β-isopropyl malate dehydrogenase inactivated Requires exogenous leucine source for growth proAB mutation...

...hypothesized that the adenosine deaminase could also perform cytidine (C) deamination, converting the C base to... picture of chromatin than an accessible/inaccessible binary. A word of caution: CGBEs and indels Cytosine...sequences. These first base editors were both deaminases: they removed an —NH2 (amine) group from the ...consisted of Cas9 fused to an engineered adenosine deaminase capable of chemically converting adenosine (A)... started engineering a CBE to be worse at its original job. They removed the uracil glycosylase inhibitor...part of the likely editing mechanism: cytidine deaminase converts C to U, and UNG excises the new U to ...could explain why the edited U base opposite the original G is not primarily converted back to a complementary...

...promoters from subretinal injections of neonatal mouse eyes at two different ages." Retinal Degenerative...optogenetics, and chemogenetics experiments. Recombinant AAVs (rAAVs) The primary application of AAV is...meaning that all viral genes except ITRs (inverted terminal repeats – the sequences that flank the deliverable...deliverable DNA) are removed, resulting in recombinant AAVs (rAAVs) with maximum “trunk” space for genetic ...placed in your cell type of interest. Careful combination of promoters, gene switches, and/or serotype ...dividing tissues will lose expression quicker than terminally differentiated, non-dividing cells.   ...size of the ssAAV genome has a mutation in the terminal resolution site (TRS) to form a vector genome ...

...homologous recombination, growing a plasmid in a bacterial strain that is deficient in recombinase protein... sequencing, (which differ from the short-read Illumina MiSeq sequencing that Addgene uses.) However, ...Plasmid multimerization occurs via homologous recombination between two or more copies of the same plasmid...plasmid (Bedbrook & Ausubel, 1976). This recombination event produces a multimeric form of the plasmid with...article.   Long-read sequencing Addgene uses Illumina MiSeq NGS to perform whole plasmid sequencing ...made with BioRender.   Grow plasmids in recombinase-deficient strains Since plasmid multimerization...bi00850a028. PMID: 4878700. Bedbrook JR, Ausubel FM. Recombination between bacterial plasmids leading to the formation...

...flexibility to the CRISPR editing world. The approach, originally published in Nature, is a “search-and-replace...HDR is typically less than 10%. In contrast, the original paper introducing prime editing demonstrated ~...into one strand, there’s a mismatch between the original sequence on one strand and the edited sequence...indels by 13-fold (Anzalone et al., 2019). PE3, as originally introduced, relies on the PE2 enzyme. PE4 and...including indels. Therefore, they engineered a dominant-negative mutant of the protein MLH1, a component...and DNA ligases to act first. PE4 and PE5, as originally introduced, rely on the PE2 enzyme. PEmax: Optimizing...(Yan et al., 2024). The team fused La to the C-terminal end of PEmax to produce PE7. PE7 significantly...

...onwards to other neuronal cells. Synapses at the terminals of axons form connections to dendrites of proximal...outlines neurons from their cell bodies to the terminals of their axons; while retrograde tracing outlines...outlines neurons in the opposite direction, from the terminals of their axons to their cell bodies. Anterograde... are some reports of bi-directionality. For predominately anterograde tracing, biotinylated dextran amines...rhodamine-isothiocyanate (RITC; Thanos et al., 1987) are used. For predominantly retrograde tracing, albumin protein labelled...AAV8, AAV9 Lung AAV4, AAV5, AAV6, AAV9 RPE (Retinal Pigment Epithelium) AAV1, AAV2, AAV4, AAV5, AAV8...conditional mutant mouse brain that expressed Cre recombinase only in pyramidal cells of the hippocampal region...

...post. From Mice to Men When this article was originally written, the main focus was on editing animal... CRISPR’s therapeutic potential in mice with a dominant cataract disorder caused by a single copy of a...edited ex vivo, and then returned to the patient. Finally, allogenic therapies involve editing healthy donor-derived...early-phase clinical trials and have shown promising preliminary results (Dimitri et al). CRISPR engineering of...and USA federal drug approval boards are being finalized, and it is very likely the treatments will be ... Cas family) as part of a cocktail therapy for urinary tract infections. The Cas3 enzymes target several...have experienced hiccups (either clinically or financially), and others we just don’t have the time to talk...

...pre-determined manner. If the original insert religates into its original vector, it will subsequently ...occasional uncut or cut/re-ligated pGGA destination plasmids. A final validation of the 5-, 12- or 24-fragment...in 5’ four base overhangs after cutting. The destination vector receives the assembly after similar Type...re-digested and "eligible” for ligation into the destination construct. Reactions are often cycled between...acquisition of antibiotic selectable markers by the destination plasmid. Antibiotic selection allowed for high...is dependent on the presence of every module, destination construct and functioning Type IIS restriction... by both advances in the re-engineering of the original BsaI-HF® Type IIS restriction enzyme and the completion...

... variety of combinations to optimize both fusion proteins. The best performing combination was NLS-dCas9...authors fragmented Cas9 into N-terminal (residues 2-713, N713) and C-terminal (residues 714-1368, C714) halves...This blog post was originally written by Caroline LaManna, published Mar 8, 2016. The updated and expanded...two fusion proteins: 1) the genomic anchor - an inactive, dead Cas9 protein (dCas9) fused to CIB1; and ...settled on a different optimal fusion protein combination. The optimized LACE system consisted of: 1) CIBN-dCas9... CIBN-dCas9-CIBN, where CIBN is the N-terminal fragment of CIB1 and it was fused to both the N- and C-...split-Cas9 (FAST) system constitutively expresses the C-terminal fragment of Cas9 while transcription of the N-...

...High-throughput cloning, in a nutshell, is the systematic combination of different genetic sequences into plasmid DNA...can be used to incorporate each element into the final construct. This strategy can be used to build vectors...availability of powerful imaging technologies, the combination of multiple fluorescent protein sequences to ...high-level expression) and a polyA signal (to terminate transcription) and the four constructs were then...create a single polycistronic 15 kb insert. The final construct was then introduced to HeLa cells (Figure...475nm Lyn-tag (derived from the tyrosine-protein kinase Lyn) Membranes 3 mCherry 587nm/ 610 nm human ...digestion followed by ligation. Upon ligation, the original pattern of the MCS is regenerated at the flanks...

...Aneesh Karve, CTO at Quilt Data. This post was originally published on the Quilt Genomics Blog and is republished...think of the human genome as a map with three coordinates: chromosome, start, and stop. For instance (chr3...as a kind of “GPS for the genome” to compute coordinates for genetic elements like protein, RNA, and DNA...DNA (Table 1). As with GPS in the real world, coordinates alone aren’t very useful. We’ll need something...come in. They help us to transform raw genomic coordinates into meaningful experiments. Table 1: An emerging...screen to disrupt these enhancers. The third and final step is to conduct our CRISPR screen. We start by...have been prohibitively difficult. CRISPR, in combination with the techniques from Table 1, provides us...

...encompass several other tag combinations, though frequently those combinations still include at least one...to aid in the expression and purification of recombinant proteins. Tags for stability and solubility ...hurdles to overcome in order to overexpress a recombinant protein? It is not generally in a cell’s best...glutathione S-transferase (GST), which can be fused to recombinant proteins for one-step purification with glutathione...for antibody-based purification; has inherent enterokinase cleavage site GST Large Protein 26 Purification...HHHHHHAGKA GEGEIPAPLA GTVSKILVKE GDTVKAGQTV LVLEAMKMET EINAPTDGKV EKVLVKERDA VQGGQGLIKI GVHHHHHH  9 Combo Consists...pure as possible? Vectors that offer different combinations of tags are readily available, and though adding...

... that utilizes phage recombination machinery to promote homologous recombination of linear DNA fragments...modified. Cytidine deaminase PmCDA1 Akihiko Kondo’s lab identified a cytidine deaminase, PmCDA1, that could...Originally published Mar 3, 2016 and last updated Apr 13, 2021 by Will Arnold. Although CRISPR systems...phage genetic systems to facilitate homologous recombination in bacteria. These quirks change the way CRISPR-mediated...selection that targets cells in which homologous recombination has not occurred. This powerful negative selection...efficiency; the only non-edited cells to survive have inactivating mutations in the Cas9 or spacer sequence, and...al., 2015). pCas contains Cas9 and the phage recombination gene lambda Red. pTargetF contains the specific...

...Originally written by Marcy Patrick and Mary Gearing on Mar 12, 2015; updated by Christina Mork, Jul ... Homologous Recombination References: Repair of Strand Breaks by Homologous Recombination. Jasin M and...DNA template used for repair is identical to the original DNA sequence at the DSB, or it can introduce specific... to as the displacement loop (D loop). The recombination intermediates can then resolve to complete the...newly synthesized sequences are retained by the original damaged DNA molecule. Unlike in DSBR, following...The size of the intended edit is the biggest determinant when selecting a type of donor. ssDNA templates...Mutagenesis (2000). PubMed. Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces...

....cell.2022.07.013 pYtags illuminate RTK signaling Receptor tyrosine kinases (RTKs) are a major class ... size (~1500 bp) makes iGECInano suitable for AAV packaging.    Find iGECInano plasmids here!   Matlashov...opening up new research directions that were inaccessible with earlier tools. They also produce more than...sensitivity, and photostability under two-photon illumination than previous GEVIs. Some constructs are also...the phosphorylated state of an immunoreceptor tyrosinase-based activation motif (ITAM). By fusing ITAM...spatiotemporal measurements of receptor tyrosine kinase signaling in living cells. bioRxiv 2022.08.13.503850....503850; doi: 10.1101/2022.08.13.503850.  iGECInano NIR calcium reporter Near-infrared (NIR) fluorescent...

...localization through different C-terminal anchoring domains The original iGluSnFR in the pMinDisplay backbone...modified form of the cytosolic C-terminal domain of Stargazin including a terminal PDZ ligand. The researchers...This post was originally written by Tyler Ford in 2018. It was updated by guest blogger Abhi Aggarwal... explore advances that have happened since our original 2018 blog post. The origins of iGluSnFR   iGluSnFR... on the extracellular side. In addition to the original PDGFR TMD, the researchers introduce iGluSnFR3...adapted from Aggarwal et al… Figure 4. PDGFR is a C-terminal fusion to the PDGFR transmembrane domain in the...expression pMinDisplay vector. GPI contains a C-terminal glycosylphostidylinositol anchor. SGZ contains...

...vivo.  Read on to learn more about this killer illumination technique! What is a photosensitizer? First...species (ROS). The ROS produced by photosensitizers inactivate target proteins or cells while having little ...within 30 minutes of exposure. 90 seconds of illumination were sufficient for 100% effective killing of...the dye. Embryos exposed to just 12 minutes of illumination had fewer mCer3-labeled cells in their hearts...photosensitizer protein that generates ROS when illuminated with green light. They found that KillerRed required...allows for precise light-based control of protein inactivation/cell ablation while having limited collateral...comments below!   References 1. He, J., Wang, Y., Missinato, M. A., Onuoha, E., Perkins, L. A., Watkins, S...

... one target is labeled and imaged at a time, inactivating the fluorescent secondary antibodies before ...comes in. After you image the first target, you inactivate the fluorophores in the sample, either by chemical...new primary, wash, add secondary, image again, inactivate, and repeat these cycles for all your targets...from. But by labeling sequentially with one, inactivating the fluorophores, then labeling with the other...brain samples from the James Trimmer Lab, using recombinant antibodies with different isotypes. (A) Neocortex...antibodies are commercially available. Pick whichever combination of host and target species, isotype specificity... 2A above, Andrews et al. used the following combination of antibodies:   Target (Rat) Primary antibody...

...piggyBac™ transposon? The piggyBac™ transposon was originally identified in moths in 1983, but wasn’t successfully...or when the gene is constitutively-active or a dominant-negative mutant. Reducing the copy number can ...host machinery for DNA repair by homologous recombination, which results in the incorporation of your ...containing a negative selection marker such as thymidine kinase, a researcher may subsequently kill off any cells...site-specific nuclease to stimulate homologous recombination and Footprint-Free™ removal of the selection...First, instead of using piggyBac™, you could use recombinase technology (Cre/loxP) to remove the donor cassette...[7], it is generally not as efficient as Cre recombinase. However, the use of negative selection largely...