...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...

...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 ...

... 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...

...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...

...interactions scientists have long used antibody based affinity purification and mass spectrometry approaches...scientists have developed newer methods termed proximity-based labeling to better capture these interactions in..., which can be a challenge for classic antibody based affinity purification/mass spectrometry methods....efficiently label neighboring proteins The first biotin based proximity labelling technique, BioID, was developed...these issues, the Ting lab utilized yeast display-based directed evolution to generate two mutants, the ...interaction detection) uses proximity protein labeling based on BioID to identify RNA-protein interactions (Ramanathan...Trinkle-Mulcahy, L. (2019). Recent advances in proximity-based labeling methods for interactome mapping. F1000Research...

... AraC transcriptional regulator Their system is based on the AraC protein that controls transcription ...pBLADEONLY: This plasmid contains only BLADE and is based on the pTRC99a plasmid. It does not include the ...the PBAD promoter. If you already have a pBAD33-based plasmid expressing your gene of interest, all you ... created by the team (Fig. 2). These images are based on the concept of bacteriographs created about a...

...which was technically challenging. Other homology based technologies We've described Sequence and Ligation...to SLIC and Gibson, there are yet more homology based assembly methods that have been described--CPEC ...store-bought cloning kits available which are all based on long-overlapping regions, have no requirements... these days. Gibson and the other long-homology based cloning methods are useful alternatives to the standard...assembly of DNA molecules up to several hundred kilobases. Nat. Methods 2009; 6, 343-345. PubMed PMID: 19363495...

...off-target edits of base editors, which convert a nucleotide base pair to another base pair at a specific...Bhoobalan-Chitty Y, Peng X (2018) Anti-CRISPR-Based and CRISPR-Based Genome Editing of Sulfolobus islandicus...CRISPR activity is an important step towards CRISPR-based therapies and for more precise editing in the lab...

...DNA sequence. Base Editors: Modified versions of CRISPR nucleases that make single base changes in the...includes the details on advances in CRISPR including: Base editing Prime editing Anti-CRISPRs … you name it...

... proteins (Cas9 shown here). The repeat sequence base pairs with the tracrRNA, and the spacer sequence...target DNA. tracrRNA: A “trans-activating CRISPR RNA” base pairs to the repeat section of the crRNA, forming...interchangeably with the term sgRNA. Seed sequence: The 8-10 bases on the 3’ end of an RNA guide (either crRNA or sgRNA...Sheet  Resources on the Addgene blog A Needle in a Base-Stack: Cas9 Structural Biology...

...the modifying sequence flanked by approximately 50 base pairs of sequence homologous to the chromosomal ... expression in yeast. These tagging vectors are based on previously described pFA6a-link vectors and include...stability, and disruption of the tagged protein. Based on their findings, the authors recommend optimal...the artifacts sometimes associated with plasmid-based overexpression. Tim Formosa, at the University of...

... Often, the choice for a fluorescent protein is based on the properties found in the literature or tabulated...is more sensible to compare fluorescent proteins based on their practical brightness in your experimental...mammalian cells this would be possible with CRISPR/Cas-based genome editing. Selecting a bright fluorescent protein...Amsterdam) where she develops genetically encoded FRET based biosensors.     Additional Resources on the Addgene...system as well as the imaging method, we recommend basing your selection on a small-scale head-to-head comparative...

... in the presence of calcium. This includes FRET-based sensors such as Twitch. While calcium is king in...Neurotransmitters Many canonical fluorescent as well as FRET-based biosensors have been engineered to detect a wide...ATP ratio. Most of these sensors are fluorescence-based and have been optimized for a wide-range of uses... pair on actinin will produce different signals based on changes to the structure of actinin in low vs...Biosensors Illuminating Epigenetics with a FRET Based Biosensor Fluorescent Proteins 101: Fluorescent...

...the first thing that comes to mind. However, ion-based communication is not just for neurons. Non-neuronal...this too. It's just that for non-neural cells, ion-based communication happens at a slightly slower scale...cases even override genetically encoded patterning. Based upon trends in studies over the past several years...Chernet et al., 2016). There are many other light-based genetic tools for studying bioelectricity in vivo...desensitization in continuous light. ArcLight Q239 - A GEVI based on the fusion of the voltage sensing domain of a...

...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...

...the field of gene therapy, more specifically AAV-based retinal gene therapy. The year was 2001 and I started...with the death of Jesse Gelsinger in an adenovirus-based gene therapy clinical trial gone horribly wrong....cell) therapies. One may wonder why the first AAV-based gene therapy treatment for a genetic disease approved...forward with ocular gene therapy The pipeline of AAV-based ocular gene therapies is impressive: clinical trials...for prevalent diseases - to name a few. CRISPR-based tools are also particularly well suited to be combined...and Wu Z., 2018. In vivo Applications of CRISPR-Based Genome Editing in the Retina. Front Cell Dev Biol...

...transcription. Sometimes enhancers are located thousands of base pairs away from the gene they regulate, but are ...detected with chromosome conformation capture (3C) based methods, such as 5C or Hi-C (Han et al., 2018). ...increase the resolution, the Xu lab created a dCas9-based CAPTURE (CRISPR Affinity Purification in situ of...DNA that are far away from each other in terms of base pairs, but close together in terms of where they...dCas9 and gRNA expression. Additionally, CAPTURE 3C-based sequencing requires a large number of cells (~5x107...Han, J., Zhang, Z., & Wang, K. (2018). 3C and 3C-based techniques: the powerful tools for spatial genome...

... sites in an orientation that results in 5’ four base overhangs after cutting. The destination vector ...IIS restriction enzyme cutting results in 5’ four base overhangs complementary to the overhangs of the ...Single insert cloning efficiencies were measured based on the acquisition of antibiotic selectable markers...markers. The 5-, 12- and 24-assembly systems were based on the ability to correctly assemble a lacI/lacZ... over 50 years, prefers ligation of Watson-Crick base pair substrates, it demonstrates significant activity...cohesive end ligation by this enzyme for all 3- and 4-base overhang sequences under standard reaction conditions...

...any plasmid-based experiment, untreated cells should be included as these provide the baseline/standard ...one thing should be present in every experiment-based investigation of a hypothesis: proper controls! ...the various controls that can be used for plasmid-based experiments. What are control plasmids? Generally...the abstract and follow an example of a plasmid-based experiment, delineate its requisite control plasmids...interpretation of the results. Typically, plasmid-based experiments employ transfection, negative, positive...completely. These controls may or may not be plasmid-based depending on the experimental needs. Using our experiment...

...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...