...biarsenical dyes such as FlAsH (green, based on fluorescein) or ReAsH (red, based on resorufin). SNAP-tag: In 2003...Johnsson’s lab developed a self-labeling fusion tag based on the human DNA repair protein AGT that gets covalently...Promega developed HaloTag in 2008 (Los et al., 2008), based on a bacterial haloalkane dehalogenase that binds...in 2008, Kai Johnsson’s lab reported another AGT-based tag named CLIP-tag, which reacts with O2-benzylcytosine...benzylcytosine(BC) ligands (Gautier et al., 2008). Although based on the same original enzyme, SNAP-tag and CLIP-tag...the other. Finding the right tag for you Protein-based tags typically provide better labeling specificity...Cornish, V. W. (2009). An In Vivo Covalent TMP-Tag Based on Proximity-Induced Reactivity. ACS Chemical Biology...

...CD20 and CD73 on specific solid as well as blood-based tumors. Monoclonal antibodies have been shown to...activated ex vivo are also contestants for cell-based immunotherapy. Using activated DCs ex vivo to generate...Recently, China spearheaded the first ever CRISPR-based clinical trial in 18 lung cancer patients. This ...differ in their genotypic and phenotypic identities based on their level of progression, anatomical location...Participants in clinical trials should be picked based on the immunotherapy target using diagnostic tests...During clinical trials, expansion of participants based on early results can enhance the positive effects...most important: How will cancers evolve and mutate based on the immunotherapeutics we use on them? Answering...

... you are trying to do: gene knockout, a specific base edit, or modulation of gene expression. Location...with BioRender.com. The jigsaw: Editing by HDR, base editing, and prime editing For a specific edit, ...constraints are even more exquisite for the so-called base editor Cas9, which makes DNA changes in the absence...dsDNA breaks (Rees et al., 2018). For C>T and A>G base editors, the intended edit must be in a 5 - 10 nt...others have examined the ability to use sequence-based and other features to nominate gRNAs that are likely...(2020) Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol...doi.org/10.1093/nar/gkw583 Rees HA, Liu DR (2018) Base editing: precision chemistry on the genome and transcriptome...

...substantial immune response or toxicity. More AAV-based CRISPR systems Smaller Cas proteins SaCas9 isn't...both is within AAV’s packaging limit. For CRISPR-based RNA editing, the REPAIR (RNA Editing for Programmable...eliminate the need to deliver larger cargo such as base editors and prime editors. While using AAV to deliver...activation. Split inteins have also been used to express base editors. References Chew WL, Tabebordbar M, Cheng...Comander J, Liu Q, Liu DR (2020) Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal.../nbt.3055  Wang D, Zhang F, Gao G (2020) CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo...

...studies of E3 ligase cereblon Optimized mScarlet-based tags for live imaging in C. elegans CRISPR/Cas9 ...proteins) and performs well in many different antibody-based applications.  IPI’s recombinant version is a fusion... O. (2024) Comparative analysis of new mScarlet-based red fluorescent tags in Caenorhabditis elegans. ...high resistance to antibiotics. Several CRISPR/Cas-based systems have been developed for other mycobacteria...and Hung, D. (2024). A dual-plasmid CRISPR/Cas9-based method for rapid and efficient genetic disruption...STReTCh (11 kDa) are significantly smaller than FRET-based mechanosensors (~60 kDa) and are compatible with...

...steel wire into a backbone structure of a protein, based on its atomic coordinates. These wire models were...momentarily forgetting that the physical model is based on their own modeling of the protein’s structure...in 2014 to ask if we could make a model of Cas9 based on the structure published by Martin Jinek’s group...recognize a PAM sequence, allow the complementary base pairing between the guide RNA and the target DNA...conformational shifts. This backbone model of Cas9 is based on Fuguo Jiang’s structure of Cas9 – primed for ...

...Kanemaki lab previously developed a CRISPR/Cas9 based method to tag endogenous proteins in HCT116 cells...sites for editing. PAM sequences can be from 2-6 base pairs in length and follow the DNA sequence targeted...SpCas9-NG) with relaxed preferences for the third base of the PAM so that it now recognizes NG rather than...of seven ChRs derived from marine microorganisms based on metagenomic data. MerMAIDs (Metagenomically discovered...

... expected sequence. Here at Addgene, we use NGS-based quality control to confirm the sequence of all the...used to confirm the rough structure of the plasmid based on the predicted sizes and organization of different...estimate how far down the gel your bands have traveled based on the dye! Always run a ladder. Ladders allow you...you get in your sample lanes. Choose your ladder based on the expected band sizes. Always run control uncut...

...spaced peaks …like these…. 3. Expect to get 500-700 bases of clean reliable DNA sequence Anything less and...uncertain terrain. 4. Never trust the first 20-30 bases of a DNA sequencing read The peaks here are usually...unfortunate side effect of messing up the reaction around base 70-75 of the read (see image below), so I would ...

...modify, whether it be editing CRISPRi, CRISPRa, or base editing. Multiplexing gRNAs: the basics One common...Gibson Assembly or SLIC cloning methods. A BsaI-based E. coli multiplexing plasmid is available from the... efficiently cleaves gRNAs sandwiched between 28 base Csy4 recognition sites. If Csy4 is not expressed...inserted into a Cas9-containing destination vector based on pCAMBIA. Cas9 is plant-optimized, and plasmids...you to assemble type II-A CRISPR arrays for dCas9-based transcriptional repression. The CRISPathBrick plasmid... method that combines CRISPR and recombineering-based MAGE (Multiplex Automated Genome Engineering) technology...only 2-3 hours incubation. Kondo Lab multiplexed base editing in E. coli The Kondo lab expressed a cytidine...

... with similar efficiency as compared to plasmid-based expression of Cas9/gRNA and can be used for most...experimental systems that may not be amenable to plasmid based methods. One of the most common techniques for delivery...gRNA RNPs to plants, as an alternative to plasmid based techniques, has been an area of intense study. Two...alleviate many issues that may arise when using plasmid based systems, most notably the ability to perform CRISPR... delivery of proteins enables efficient protein-based genome editing in vitro and in vivo." Nature biotechnology...

...has been started based on DETECTR technology with the mission to “provide a CRISPR-based platform on which...SHERLOCK and DETECTR demonstrate the power of CRISPR-based diagnostics. SHERLOCK- Specific High Sensitivity... DNA or RNA with atomolar sensitivity and single base pair mismatch specificity.  SHERLOCKv2 SHERLOCK ...isothermal amplification by RPA. When Cas12a-cRNA base pairs with the dsDNA of interest, the DNase activity...Wadford DA, Chen JS, Chiu CY (2020) CRISPR–Cas12-based detection of SARS-CoV-2. Nature Biotechnology. https...

...optogenetics, such as channelrhodopsins and tools based on light-oxygen-voltage (LOV) domain. They can also...interactions, we recommend using NIR split reporters based on miRFPs (Shcherbakova et al., 2016), since they...Filonov et al., 2013). FRET biosensors were developed based on miRFP760-miRFP720 FRET pair, including Rac1 biosensor...intensiometric calcium indicators NIR-GECOs were developed based on mIFP6. Learn more about the characteristics, ...Brightness in HeLa cells vs. iRFP713, % d Ref BphP-based FPs miRFP670 https://www.addgene.org/79987/ 642...720 98,000 6.1 97 Monomer 510 4.5 117 12 CBCR-based FPs miRFP670nano https://www.addgene.org/127443...

...different parameters that your gating strategy will be based upon. We’ll try to keep it short though, promise...SSC: scattering To digitally extract your cells based on their size, the cytometer provides you with two...lymphocyte population (10.7% of all recorded events) based on their expected size. The strong signals close...not of interest can still be gated out later. Based on personal preference, some people like to make...staining signals. For this, you’ll need to gate based on the proportion between H and A (or W and A) within...When you’re interested in measuring a population based on a fluorescent stain, you might be wondering which...

...system. Biosensors based on Nano-lantern technology are also very robust, with an eNL-based calcium sensor...RLuc8) with Venus. This luminescent technology is based on the principle of bioluminescence resonance energy...They also developed calcium, cAMP, and ATP sensors based on Nano-lantern, showing the technique’s versatility...

...time was not right for the development of phage-based therapeutics. After a brief stint at the venture...from developing therapeutics to developing phage-based tools for detecting pathogens in food products. ...biotechstart.org Read Sample 6's Paper on Bacteriophage-based Diagnostics Additional Resources on the Addgene...

... let the virions out. We recommend using a Tris-based lysis buffer and sonication.  The obtained virions...particles that can transduce cells, which can vary based on target cells and can be altered by freeze/thaw...experimental outcome and planning.  Both ddPCR- and qPCR-based titering assays measure physical titers, meaning...post.  We generally do not recommend using ELISA-based titering assays, since those will quantify both ...Inactivation of Adeno-Associated Viral Vectors by Oxidant-Based Disinfectants. Human Gene Therapy, 32(13–14), Article...

...are many protein-based toxin-antitoxin systems. One of the most well known protein-based toxin-antitoxin...counter this, methyltransferases add methyl groups to bases at specific nucleotide sequences, preventing the...system that is regulated by antisense RNA, which base pairs to cognate mRNA to inhibit its translation...Crystal structure of the MazE/MazF complex: molecular bases of antidote-toxin recognition." Molecular cell 11.4...

...gravity-based column plasmid maxiprep kit takes 1-1.5 hours to complete, while this syringe-based method...Compared to a commercial magnetic bead-based kit, this cellulose-based method was faster, cheaper, and had... Addgene follows a similar workflow as a column-based plasmid extraction kit. First, you lyse the bacteria...

...for studying colloidal gold (1).  The method was based on using a thin plane (sheet) of light generated...microscopes, optical sectioning of a specimen is based on discriminating the out-of-focus reflected light... combining Ultramicroscopy with tetrahydrofuran-based tissue clearing (13,14). A few years later, Karl...Deisseroth’s lab developed CLARITY, which is a hydrogel-based method that also allows antibody penetration for...recently developed AAV (adeno-associated virus)-based fluorescent labeling methods that can be used with...