.... Development of this tool required creating a database of common features, and devising rules for identifying...when the match is imperfect. SnapGene's feature database The source of common features was our collection...issue is addressed by adding more variants to the database. Another limitation is that by tolerating mismatches.... To prevent misidentification, we augment the database with new fluorescent protein variants. The future...features? We plan to continue updating the feature database. Powering the plasmid maps on Addgene.org with... which common features need to be added to the database – we will work to fill any common annotation gaps...SnapGene supports “Collections”, which are shareable databases of DNA and protein sequences, the next step is...

...of research into COVID-19. While this program is based in Canada, much of the information is applicable...support this pace of research, new, centralized databases and centers to support the sharing of materials...into COVID-19 (like here at Addgene!), and new databases are supporting their access to useful resources...internationally COVID-19 Resources Canada is a database that provides information about reagents, funding...the future of research. To find other COVID-19 databases and resources, check out our growing Twitter thread...

...Coselection with Ouabain New CRISPR base editors The popular CRISPR base editing technique uses dCas9/Cas9...specificity of base editing. Kim et al. used natural and engineered Cas9 variants to develop five new base editors...number of available target sites for base editing. For each base editor, they observed editing activity...cytidine deaminase portion of the base editor to create SpCas9 base editors with editing windows as small...off-target effects associated with base editing, Rees et al. created HF-BE3, a base editor containing high fidelity...diseases. The Tol2 Gateway-Compatible Toolbox is based on the original Tol2kit generated by the Chi-Bin...These exciting papers further show the potential of base editing for precise genome modification. Plasmid...

...increasing CRISPR editing efficiency, new CRISPR base editors, DIY DNA ladders. You can find additional...Segement on Ouabain Selection  Hot Plasmid 3: CRISPR base editors with enhanced range and specificity. Listen...Listen to to the Segmennt of Improved CRISPR Base Editors Hot Plasmid 4: pPSU Plasmids for generating DNA...

...ex: genome editing), our MUCICAT system which is based on the CRISPR-associated transposase system, INTEGRATE...gene expression is an alternative versus plasmid-based expression which can be unstable and lack precise...application of a multiplex genome editing system based on CRISPR-associated transposition.     References...

... editing (SECURE) base editors Keith Joung’s lab recently found that a cytosine base editor (CBE) with...Covalent tethering of DNA template to Cas9 SECURE base editors Nme2Cas9 CRISPR interference in Candida ...efficiency. Read the paper in Nature Find the SECURE base editors All-in-one AAV delivery of Nme2Cas9 with...

.... “You can think of changing bases to other RNA bases or unnatural bases,” she said. “The possibilities...modular repeats, each of which binds a consecutive base in its RNA target. By changing amino acids, it’s...

...initially recognized as a useful vertebrate model based on their straightforward husbandry and ability to...developmental biology that arose from zebrafish-based investigations. Genetic resources for zebrafish...important insights in developmental biology. Microscopy-based lineage tracing Over a period of 24 hours, the externally...approaches (Keller et al., 2008). This microscopy-based lineage tracing defined a high-resolution map of...from the random mutagenesis screens. Microscopy-based lineage tracing can help illuminate how signaling...during embryo development. Single cell sequencing-based lineage tracing Recently, gene expression in all...rather than novel, abnormal cell types. Sequencing-based lineage tracing can help elucidate the gene networks...

...overview of the general schematic of CRISPR/Cas based COVID-19 testing methods.   Rapid detection of...2019 novel coronavirus SARS-CoV-2 using a CRISPR based DETECTR assay Sensitivity:10-100 copies of RNA per... al., 2020). Detecting coronavirus using CRISPR-based SHERLOCK (Specific ​H​igh ​s​ensitivity ​E​nzymatic... A scalable, easy-to-deploy, protocol for Cas13-based detection of SARS-CoV-2 genetic material (CREST)...on the target regions thus making it an affinity-based method. Due to the mismatch sensitivity of FnCas9...Wadford DA, Chen JS, Chiu CY (2020) CRISPR–Cas12-based detection of SARS-CoV-2. Nature Biotechnology. https...coronavirus SARS-CoV-2 sequence detection method based on CRISPR-Cas12. Cold Spring Harbor Laboratory. ...

...be used for cloning or genome engineering and is based on homologous recombination. It allows for direct...non-selectable sequences, point mutations or other small base pair changes, and the addition of protein tags. ...best for point mutations or changes of only a few base pairs.  dsDNA Substrate dsDNA inserts can be made...the DNA sequence of interest and flank it with 50 base pairs of homology to the targeted insert site (Figure...way is to introduce a C/C mismatch at or within 6 base pairs of the edit site. This is an easy fix, but...the wobble codons, i.e. make changes to the third base pair of the adjacent 4-5 codons that alter the nucleotide... DL. Recombineering: A Homologous Recombination-Based Method of Genetic Engineering. Nature protocols....

... the Cameleon-based FRET sensor D1ER, and GCaMPER. Bonus: There are also Cameleon-based FRET sensors available...kinetic parameters. There are three GCaMP types based on their kinetics: GCaMPf (fast) GCaMPm (medium)...wavelength is insensitive. Additional red sensors based on mRuby (jRCaMP1) and mApple (jRGECO1a) are also...reduce these unwanted variables. Twitch is a FRET-based calcium reporter derived from troponin C. It is ...mechanism-guided design of single fluorescent protein-based biosensors. Nature chemical biology. 2021 May;17...

... of viral particles in a sample and are usually based on the presence of a viral protein, such as p24,...incorporated into a viral particle. Consequently, titers based off of p24 quantification tend to be overestimates...depending on the vector backbone. When using qPCR based methods for viral titration consider using a reverse...by antibiotic-resistance or single cell sorting based on expression of a surface marker, subcloning, expansion...more straightforward approaches such as the FACS-based or colony-forming unit assays, whereas labs that...concentration and titration of pseudotyped HIV-1-based lentiviral vectors."Nature protocols 4.4 (2009):...

...is another very experienced and successful coach based in New York. Another experienced coach and speaker...you. There is also a group The Dissertation Coach based in Illinois. A few of the coaches in this group ...specialty for Michelle Capes at Adeptify Me. While based in Madison, Wisconsin she also works remotely and...recruiter. Chris Humphrey from Jobs on Toast is based in the United Kingdom and scientists there would...content for PhDs and postdocs posted by Toronto-based Jennifer Polk of From PhD to Life. She runs great...interesting and it is in English and Spanish. She is based in Spain. The bottom line – there are lots of people...

...fluorescently labeled probes. Colorimetric and luminescence based detection methods are also commonly used. In all... Since the sequencing gel will separate the DNA based on size, the resulting gel should ultimately contain...correspond to every nucleotide in your DNA sequence, based on which ddNTP was added and when. Compiling the...fluorescently labeled ones allowing for fluorometric-based detection. This increased sequencing speed ultimately...world, one up and coming innovation is nanopore-based DNA sequencing which reads the nucleotides of a ...nucleotides (8). Compared to older technologies nanopore-based DNA sequencing machines are relatively small meaning...error rates (10). It is projected that nanopore-based sequencing technologies could one day sequence an...

...and image for long periods of time.  Are antibody based probes a solution? To address these limitations,...these to tag their proteins of interest. Antibody based probes, such as antigen-binding fragments (Fabs)...frankenbody To combat the issues with these antibody based probes the Stasevich lab used an alternative strategy...strategy to develop a genetically encoded antibody based probe that detects the widely used HA epitope in...CDR grafting to stable frameworks and structure-based framework engineering. Methods, 34(2), 184–199. ...

... types Must be verified with cell-based method IDLV 1% Cell-based Programmable, can detect DSBs in live... genome and pinpoint the location of mismatches based on the sequences of the genome and sgRNA. This is...have disastrous health effects in the clinic. NGS-based methods provide the most complete information profile...al. 2016) and HTGTS (Frock et al. 2015) for cell-based experiments. These strategies can be used in concert...off-target editing events. Two of the most common cell-based methods are genome-wide, unbiased identification... Advantages Disadvantages GUIDE-Seq 0.1% Cell-based  Searches the genome for all DSBs, doesn't require...methods, high background BLESS Not reported Cell-based (in vitro) Can be used on tissue from whole animal...

...George Abraham, Bobin., et al. "Fluorescent protein based FRET pairs with improved dynamic range for fluorescence...voltage sensors), to name a few.   Find Many FRET-Based Biosensors on Addgene's Biosensor Pages   Recent...Atsushi, et al. "Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin." Nature... Fanjie, et al. "A fluorescence energy transfer‐based mechanical stress sensor for specific proteins in.... Gayrard, Charlène, and Borghi, Nicolas. "FRET-based molecular tension microscopy." Methods 94 (2016)...about Fluorophore Targeting for FRET Studies FRET Based Biosensors Additional Resources on Addgene.org ...

...system. Site Directed Mutagenesis by PCR & Single Base Editing with CRISPR For my editor’s choice picks...picking two posts that describe methods to make single base edits in DNA. The first, contributed by guest blogger...Scientist Mary Gearing and discusses a newer, CRISPR-based technique to generate C->T or G->A transitions in...Tips for Troubleshooting Viral Transductions Single Base Editing with CRISPR Cas9 Activators: A Practical...

...least ~200 base pairs of sequence flanking the editing site on either side of the amplicon. Based on the ... A specific insertion or deletion? A knock-in? A base edit? Once you know exactly what you are looking...repair events, or maybe you’re performing a single base pair edit with a prime editor. What next? When generating... you introducing a large insertion, something 20 base pairs or bigger? Then screening by PCR may be the...knock-ins Is your knock-in edit small, with only a few base pairs modified? In these cases, it’s best to do ...Restriction enzyme screening is sensitive to single base pair changes, but the edit must occur at an existing...

... et al., 1998). There are two models for iteron-based plasmid replication control that are not necessarily...synthetic biologist who wants to develop a plasmid-based tool that is easy to use with other plasmids.  How...developed a system of bacterial incompatibility groups based on similarity of replication and partitioning systems...determine how compatible their plasmids will be based on sequence data. However, if these do not provide... antimicrobial resistance. Using novel plasmids based partly on plasmid incompatibility with antibiotic-resistance... R, Rech J, Bouet J-Y (2015) Imaging centromere-based incompatibilities: Insights into the mechanism of...microbial plasmids: classification and identification based on replication and transfer systems and host taxonomy...