... NIH-sponsored effort to provide gene-editing nucleases to the zebrafish community. Target Gene Name TALENs...

...through Addgene. Visit our viral service page to learn more about our viral service, and see which plasmids...

...e.g. converting a DNA binding protein to a DNA cleaving enzyme) or to regulate the activity of a protein...

...://doi.org/10.1089/hum.2019.277 Lavin TK, Jin L, Lea NE, Wickersham IR (2020) Monosynaptic Tracing Success...BioRender.com   Causes of recombination and leaky expression While leaky expression and spontaneous recombination...will have some level of recombination that can lead to leaky expression in cells that do not express Cre ...scrambled cre-recombinase sites lead to greater frequency of reversion and leaky expression. On the right non-homologous...recombination between these sites may be the cause of leaky expression. To test their hypothesis, they randomly...recombination. This indicates that the amount of leaky expression can largely be attributed to the level... non-homologous scrambled recombination sights lead to reduced reversion and less off-target expression...

...RuvC-like endonuclease domain, but they lack Cas9’s second HNH endonuclease domain. Cpf1 cleaves DNA in ...orthologs that display cleavage activity in mammalian cells. Like Cas9 nucleases, Cpf1 family members contain...based system, require a single-component nuclease to mediate cleavage rather than the multi-subunit complex...contains a RuvC-like endonuclease domain, but it lacks Cas9’s other HNH endonuclease domain, indicating ...functional CRISPR nuclease that could be adapted for genome editing. The use of a different nuclease could potentially...that display robust cleavage activity similar to that of Cas9. These two nucleases, AsCpf1 and LbCpf1 ...proximity to the cleavage site, preventing future edits. In contrast, since Cpf1 cleaves relatively far ...

...Cas13a. The origins of Cas13a: An RNA cleaving CRISPR nuclease Cas13a was originally identified in 2015... crRNA to cleave target DNA (Cpf1 requires only a crRNA for target recognition and cleavage). C2c2 (now...domains to cleave target DNA. The HEPN domains within Cas13a are essential for RNA cleavage, consistent...bind and cleave RNA non-specifically. The ability of dCas13a to bind target RNA without cleaving the molecule...between Cas13a and Cas9 is that Cas13a binds and cleaves RNA rather than DNA substrates. In terms of structure... residues in the Cas13a molecule results in a “nuclease dead” Cas13a (dCas13a), that is capable of binding...binding target RNA but lacks the ability to cleave the RNA target.  Table 1: Comparison on Common CRISPR...

...gets activated. This unleashes indiscriminate ssDNA cleavage activity which cleaves the supplied fluorescently...fluorophore that is quenched when intact and released when cleaved. Cleavage results in fluorescence and this approach... This activation results in a trans cleavage reaction that cleaves a nearby RNA sequence that contains...activated and performs non-specific collateral cleavage. This cleavage is coupled to fluorescence visualization...fluorescently labelled ssDNA substrates. Upon cleavage, the fluorescence is no longer quenched and can be ... a PAM (Protospacer Adjacent Motif) for target cleavage. This test can be performed in two steps for challenging...ssDNA-FQ reporters that exhibit fluorescence upon cleavage. Upon amplification by the RPA primers, the Cas12a-crRNA...

...) to cleave ssRNA or mRNA. It also exhibits a “collateral effect” of non-specific ssRNA cleavage. How ...ssRNA cleavage byproducts. Thus, binding of Cas13 to the programmed region of interest would lead to both... the Addgene Blog Learn about using toehold switches to detect Zika virus   Learn more about targeting...degradation to develop the method termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) (Chen...Both SHERLOCK and DETECTR harness the promiscuous cleavage and degradation of neighboring ssRNA and ssDNA...ssDNA by Cas13 and Cas12a, respectively, to cleave and activate a reporter. The detectable signal from this...to the programmed sequence, it can promiscuously cleave surrounding ssRNA molecules. In SHERLOCK a quenched...

...for Cas9 nuclease function. Stem loops 2 and 3 are not strictly necessary for in vitro cleavage, but they...the two nuclease domains, Cas9 ensures both are positioned correctly before HNH or RuvC can cleave DNA, ...? It’s not actually clear! In vitro, Cas9 holds tightly to DNA even after cleaving it, so many researchers...profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature Biotechnology...   Cas9 has two major lobes, called NUC (for nuclease) and REC (for recognition). The NUC lobe contains... and RuvC domains, which are named for similar nuclease folds found in other proteins. The C-terminal ... and helps recognize the guide RNA. Unlike the cleavage domains, the recognition domains—including the...

... strands hybridize, the nuclease can detect strands with mismatches and cleave them. The results are then...surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases." G3: Genes| Genomes...profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity." Nature biotechnology...genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases." Nature biotechnology 33.2 (2015)...genotyping. The most common options include mismatch cleavage assays, such as Surveyor™, T7E1, and Sanger sequencing...adequate standards for validating edits.  Mismatch cleavage assays rely on pairing between the edited strand... in the genome predicted to contain off-target cleavage events. Unbiased techniques search the whole genome...

...sequence! In bacteria, Cas13d-mediated cleavage promotes collateral cleavage of other RNAs. As with other Cas13s...and two nuclear long non-coding RNAs. One interesting twist to Cas13d enzymes is their cleavage pattern...EsCas13d favors cleavage at uracils, but a more detailed exploration of this cleavage pattern is necessary...a CRISPR repeat array near a putative effector nuclease. The Cas13d proteins they identified have little...identified Cas13a-c orthologs, but they do include HEPN nuclease domains characteristic of the Cas13 superfamily...their own CRISPR arrays into guide RNAs. crRNA cleavage is retained in dCas13d and is thus HEPN-independent...Cas13s, this collateral cleavage does not occur when Cas13d is expressed in a mammalian system. Since Cas13d...

...and structural integrity; cleavable. Not great for purification; too cleavable in eukaryotes TAP GRRIPGLINP...FLAG) are cleaved by specific proteases without requiring the addition of an independent cleavage recognition...cell. FLAG tags can be cleaved by enterokinase, which recognizes DDDDK^X, cleaving after the lysine. The...Notes TEV ENLYFQS Cleaves between the Gln and Ser residues Thrombin LVPRGS Cleaves between Arg and Gly...maltose-binding protein (MBP). Overexpression can also lead to insolubility, and aggregated protein is not useful...sequence of amino acids, is basically a molecular leash for your protein. If you’re working with an uncharacterized...-based purification; has inherent enterokinase cleavage site GST Large Protein 26 Purification and Stability...

...characterized two Cpf1 nucleases, distant cousins of well-known Cas9. Cpf1 cleaves DNA in a staggered pattern...detectable off-target cleavage. Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites...proximity to the cleavage site, preventing future edits. In contrast, since Cpf1 cleaves relatively far ...Cpf1-mediated cleavage, the continued presence of the PAM may give Cpf1 the ability to cleave again and possibly...the two (tracrRNA and crRNA) needed by Cas9 for cleavage. Now, two new studies show that Cpf1 displays ...blunt ends 3 nt upstream of the PAM site, Cpf1 cleaves in a staggered fashion, creating a 5 nucleotide...18-23 bases away from the PAM. Cpf1’s staggered cleavage pattern opens up the possibility of directional...

...have two endonuclease domains: the n-terminal RuvC-like nuclease domain and the HNH-like nuclease domain...conformational change that positions the nuclease domains to cleave opposite strands of the target DNA. Thus...and Cas9, bind to these transcripts and C) cleave them leaving mature crRNAs bound to the Cas9/tracrRNA ...Cas9 complex to the target DNA which is E) cleaved leaving a F) double-strand break. A “gRNA” is a researcher-designed...Cas9 genome editing systems use a non-specific endonuclease (Cas9 or closely related Cpf1) to cut the genome...genome and a small RNA (gRNA) to guide this nuclease to a user-defined cut site. After reading this post...functions of the various CRISPR/Cas9 components. Please note that while this post is intended to provide...

...every team should have a goal, or at the very least, a clear reason for existing. Here are a few types of...own experience: Leadership Few things are more frustrating than being on a leaderless team. Sometimes ...team, I try to make it very clear why that person is there. If it becomes clear after a few meetings that...strong sense of purpose, an excellent leader, and perfectly clear lines of communication, but if teammates...this gap by both highlighting that researchers do learn MANY transferable skills while working in the lab...The famous discoveries and experiments I’d been learning about for years were usually the products of teams...forward nearly 20 years and I am known by some colleagues as the “cross-team guy” at Addgene, an unofficial...

...serve as slaves.  Meganucleases In 1989 the first ever DNA-editing enzymes, meganucleases, were discovered...created by genetics.    Zinc finger nucleases Zinc finger nucleases (ZFNs) are artificial restriction enzymes...DNA-binding domain to a DNA-cleavage domain (FokI). Zinc finger nucleases were first designed in 2005....activator-like effector nucleases (TALENs) were engineered. TALENs follow a DNA-binding and DNA-cleaving strategy similar...The major drawback of all the endonuclease-based gene editors (meganucleases, ZFNs, TALENs, and CRISPR-Cas...2002) Mutations altering the cleavage specificity of a homing endonuclease. Nucleic Acids Research 30:3870...in 1990, just a year after the discovery of meganucleases. The storyline involves the resurrection of ...

...these authors were able to reconstruct a cleared guinea pig cochlea in 3D. The basic configuration of the...passes through the specimen, which often leads to photobleaching.  Furthermore, point-by-point scanning ... of clear specimens and for in vivo imaging of transparent organisms. Advances in tissue clearing Similar...modified by others to produce Murray’s clear solution.  Murray's clear (11) was mostly used to study the development...with diameters less than 4nm. Lateral view of a cleared rat pons, medulla and the first segments of the...fluorescence microscopy was first pioneered by Voie and colleagues and originally named orthogonal-plane fluorescence...reconstruction (5). In order to image large tissue such as cleared rodent brain, the Ultramicroscope was developed...

... follow a lead and when to terminate a project are fundamental skills that all group leaders must master... 8. Learn to mentor, manage, communicate, and teach The dirty secret about becoming a group leader is ...teaching experience and learn about modern pedagogy techniques, especially active learning. Serve as a daily...Assuming you want to become competitive for a Group Leader position, what will you need? Multiple first ...universities and institutes annually advertise for Group Leader positions starting in August, the year before the...completed. Therefore, to be ready to apply for a Group Leader position, key first author manuscripts need to ...perspective for your project. Furthermore, Group Leader position applications require 3 letters of reference...

...Cpf1 CRISPR Nucleases   Cas9 Cpf1 Components crRNAtracrRNACas9 crRNACpf1 Endonuclease size spCas9... multiple targets compared to Cas9.  Read on to learn more about Cpf1 multiplexing.  For an in-depth review...crNA: ~42 nt Requires tracrRNA? Yes No dsDNA cleavage Blunt end 5' overhang PAM sequence NGG TTTV ...PAM site preserved? Usually Destroyed Yes, Cpf1 cleaves 5' of the protospacer Multiplexing CRISPR-Cas9...express the gRNAs and Cas9. Co-transfections can lead to variable expression levels due to differences...allow for expression of multiple gRNAs (i.e. Csy4 cleavage sequence, tRNAs, multiple individual promoters...requires its own promoter   Kabadi et al Csy4-Cleavable Cassettes transfection gRNAs expressed from a ...

...multiplexing gRNAs used the RNA nuclease Csy4 from Pseudomonas aeruginosa to cleave 4 gRNAs separated by 28 base...cell, this system is clearly also robust enough to ensure high levels of gRNA cleavage.   Xie et. al constructed...sequences preceding gRNAs, to test for gRNA cleavage. gRNAs were cleaved precisely as predicted with no addition...schematics. Scissors indicate cleavage sites. After confirming gRNA cleavage, they examined gRNA expression...with Cas9 and the polycistronic transcript. As nucleases may have off-target effects and/or toxicity, it...every living organism. Precursor, or pre-tRNAs are cleaved by RNase P and Z in eukaryotes (E in bacteria),...elements of tRNAs are necessary for recognition and cleavage. This sequence flexibility indicates that gRNAs...