...situ of Regulatory Elements: The original CAPTURE method The original CAPTURE method requires the creation...Originally published Dec 7, 2017 and updated Jul 2, 2020. Promoters may be the star of gene regulation...Purification in situ of Regulatory Elements) method. The original CAPTURE method was published in 2017 and addressed...by PCR or deep sequencing. A drawback of the original CAPTURE method is that a new cell line has to ... of the CAPTURE 2.0 system To improve upon the original CAPTURE method, the Xu Lab made the following ...changes to create CAPTURE 2.0:  Replace the biotinalatable tag on dCas9 with a BioTAP-tag. The BioTAP-...delivery of the two remaining CAPTURE 2.0 components eliminates the need to create stable cell lines and allows...

...peptide, while three bound to the C-terminal peptide. One of these C-terminal antibodies was Myc1-9E10 (aka ...Munro S, Pelham HRB (1987) A C-terminal signal prevents secretion of luminal ER proteins. Cell 48:899–907...Tagging Plasmid-based Recombinant Antibody Resources on Addgene.org  Find recombinant anti-c-Myc [9E10] ..., 1984). The strategy they landed on was to recombinantly append their proteins of interest with unrelated...protein had a highly specific antibody to the C-terminal five amino acids, meaning the tag could be quite...). They noticed that Myc1-9E10 and the other C-terminal antibodies only recognized the human c-Myc protein...sequences in these species, they found that the N-terminal half of the antigen differs significantly between...

...through site-specific recombinases such as Cre or Flp that induce DNA recombination at defined recognition... with an inactive opsin gene; in Cre expressing cells, however, Cre induces the recombination of the double-floxed... P1 site-specific recombination. Purification and properties of the Cre recombinase protein." Journal ...Taylor et al., 2016; Hooper and Maguire, 2016). Recombinant adeno-associated viruses (rAAVs) have become ...opposite orientations. Since Cre does not cause recombination between mismatched recognition sites, its presence...viruses, such as the optogenetic FLEx vectors in combination with transgenic animals or rAAVs expressing Cre... "Adeno-associated virus 2/9 delivery of Cre recombinase in mouse primary afferents." Scientific reports...

...thereby rendering them inactive in the cytoplasm. The authors use PhoCl in combination with this caging technique...Tol2 Gateway-Compatible Toolbox is based on the original Tol2kit generated by the Chi-Bin Chien lab (Kwan...with nonoverlapping emission spectra (N- and C-terminal tags for mTagBFP, TagRFPt, EGFP, mVenus, mCerulean3...Gateway-Compatible Toolbox Podcast Segment The impact of KLF4 N-terminal variants on iPSC generation Deriving iPSCs (induced...discrepancies, which they traced to a 9 amino acid N-terminal variation in the Klf4 isoform (KLF4s or KLF4L)...variant they call mScarlet. Bindels et al used a combination of deliberate and random mutagenesis to isolate...light-dependent Gal4 transcriptional activation and Cre recombination. Find PhoCl plasmids at Addgene. Zhang W, et...

...today's biological research requires a close examination of specific proteins within a system. This can...expressed in frame on the N- or C-terminus of a recombinant protein of interest. The tag was developed as...wash steps removes excess proteins and other contaminants. The protein of interest is then eluted using...tagged protein of interest (purple) and several contaminating proteins (blue, green, orange) flows through...immobilized along with a polyhistidine containing contaminating protein (orange). A low imidazole wash buffer...buffer disrupts the weak interaction of the contaminating protein but not that of the protein of interest...wash buffer should be high enough to disrupt contaminant-metal ion binding without eluting the target ...

...cabinet, it needs to be decontaminated in a biosafety cabinet. When decontaminating liquid waste, remember...production), there is a significant risk for aerosol contamination. As most centrifugation has to be done outside...heat sealed tubes can be used for extra security. Finally, when spinning down lentivirus, you’ll need to ...you should open in the biosafety cabinet and decontaminate after use. Virus-exposed plastics and other ...other surfaces should be thoroughly decontaminated after use, typically with bleach (plastic) or ethanol (...cities may require a higher percentage of the decontaminate to be used for BSL-2 waste, and of course, if... percentage indicates the proportion of the decontaminate in the waste itself, not in the solution that...

...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...