... created a codon adaptation index (CAI) that is based on the codon usage frequency in a reference of highly...improve gene expression by changing synonymous codons based on an organism's codon bias. The idea is that scientists...can make mutations throughout a gene of interest based on an organisms codon usage bias to increase translational...code (Gustafsson et al., 2004), such as Hoogsten base pairs and wobble pairs.  Figure 2: (A) The...different tRNAs, including those that exhibit wobble base-pairing (a tRNA that can recognize multiple synonymous...Chappell SA, Zhou W, Mauro VP (2005) An mRNA-rRNA base-pairing mechanism for translation initiation in ...-0270(02)00362-x Stadler M, Fire A (2011) Wobble base-pairing slows in vivo translation elongation in ...

...Class 2 CRISPR systems, including the type II Cas9-based system, require a single-component nuclease to mediate...fashion, creating a 5 nucleotide 5’ overhang 18-23 bases away from the PAM. With this information, they turned...advantages of Cpf1 over Cas9 Type II CRISPR systems based on Cas9 were thought to be the simplest CRISPR systems...with single and double mismatches throughout the 23-base sequence. Double mismatches ablated Cpf1 activity...were present in the 3’ end of the target sequence (bases 19-23). Cpf1 is also sensitive to single mismatches...et al. saw no decrease in Cpf1 activity with 4-6 base deletions at the 3’ end of the target sequence. ... Figure 2: Effects of (A) double and (B) single base pair gRNA-target mismatches on modification by AsCpf1...

...previously uncharacterized enhancers. The popular TALEN-based LITE system, which uses light to regulate transcription...deacetylases. CRISPR and epigenetics As with many TALEN-based technologies, the advent of CRISPR has made targeting...strand breaks. Below you'll find some of the CRISPR-based epigenetic modifiers available from Addgene. For...Albert Cheng’s lab developed Casilio-ME, which is based on their Casilio system. This system allows for ...Epigenetic modification is certainly not the only CRISPR-based technology designed to alter gene expression. Fusing...cloning platforms for transgene expression and dCas9-based epigenome editing. Nucleic Acids Research 47:e23...Gersbach CA (2015) Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters...

...vectors and a reporter vector for mammalian cell-based validation assay that are designed for use with ...through in vitro transcription as an alternative. Based on transient double-selection of transfected cells...TALEN kit. This backbone is suitable for the TALEN-based generation of germline mutations in Bombyx mori ...

...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...

.... 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...

...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...

...Browse CRISPR Plasmids By Function Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation ... your system, you can search or sort this table based on: Your particular expression system , eg. Mammalian... and FokI-dCas9. Yamamoto Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector...System Mammalian MultiSite Gateway recombination-based cloning of complex polycistronic lentiviruses. Can...Genome Editing in Monocot and Dicot Plants Plant PCR-based procedure to rapidly generate multiple sgRNA expression...available for use in this Golden Gate and Gateway based assembly method: plasmids for assembly of single... cassettes using Golden Gate ligation into Tol2-based pGGDestTol2LC vector. Uses five distinct zebrafish...

...off-target effect). The double nickase system is based on the Cas9 D10A nickase described in Figure 4 of...step-wise instructions on designing and using Cas9-based genome engineering tools to perform different types...express without the G. Because the G is only one base, the Zhang lab usually adds it when they order the...oligo from IDT (non-PAGE purified oligo is fine based on new data). For general transfection, you could...

... 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...

... allows you to isolate and purify DNA fragments based on size. The procedure starts with standard agarose...electrophoresis, which separates DNA by their length in base pairs. Following electrophoresis, you can cut DNA...molecular cloning, such as PCR - or restriction enzyme -based cloning. Protocol: Gel Purification Follow the Agarose...

...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...