...and responsive voltage indicator with enhanced sensitivity for unitary synaptic events By Brian O’Neill ...ASAP5 selected for its improved kinetics and responsivity in screening ASAP3 variants. B) Electrical and... and optical recordings of spontaneous activity in human stem cell-derived neurons expressing ASAP5-Kv...regimes with fast activation kinetics and high responsivity near the resting membrane potential, ASAP5 is...and responsive voltage indicator with enhanced sensitivity for unitary synaptic events. Neuron, 112(22),...context-dependent responses and molecular patterns of selectivity. With 10 plasmids available as a kit, plus 21...Direct interrogation of context-dependent GPCR activity with a universal biosensor platform. Cell, 187...

...gRNAs in vivo, they should be screened for activity in vitro and then packaged into AAV for in vivo delivery...SPH mice CRISPRa has largely been limited to in vitro settings due to the large size of Cas9 activators...developed neuron-optimized lentiviral vectors for in vitro and in vivo CRISPRa (Savell et al., 2019). This ...Bdnf is expressed early in response to neuronal activity (hence, “immediate-early”) and consequently regulates...immediate-early genes associated with neuronal activity, such as Fos, Egr3, and Arc. Thus, CRISPRa represents...preferable for individual gene activation in select in vitro and in vivo applications, the tools described above...

...biosensor has a high sensitivity (50 nM-500nM range) and a high specificity for SLs in vitro, and they further...SpCas9 variants have improved activity on NAG PAMS and reduced activity on NGG PAMs relative to wild type...mimic aspects of organ morphology and physiology in vitro and have emerged as a powerful model for understanding... the enzymatic biosensor retains its catalytic activity.  As SLs are also known to trigger seed germination...

...phosphorylated DNA termini to 3 ́-OH DNA termini (1-4). In vitro, ligases (notably T4 DNA ligase) are critical reagents...circularization of dsDNA (6). Less commonly utilized in vitro, Taq DNA ligase will ligate only nicks (5-8). Taq... applications, there cannot be any end-joining activity, and for others, there is a need for the exclusive...or qPCR-based methods to greatly increase the sensitivity of detection (14-20). LDR has also been extended...achieved through a combination of the intrinsic selectivity of the ligase active site and careful balance... that extends earlier studies through a high-sensitivity multiplexed format (21, 27). This methodology...Bruce Wallace. "Specificity of the nick-closing activity of bacteriophage T4 DNA ligase." Gene 76.2 (1989...

...Provided 5% Inexpensive, simple Low-throughput, low sensitivity Sanger Sequencing Provided 20% (variable) Sequence-level...inappropriate for heterogenous populations, low sensitivity Targeted Amplicon Sequencing Provided 0.01%...including Digenome-seq (Kim et al. 2015) for in vitro analysis, IDLV for in vivo detection (Gabriel et...potentially toxic) Digenome Seq 0.1% Cell-free (in vitro) Works across all cell types Must be verified with... background BLESS Not reported Cell-based (in vitro) Can be used on tissue from whole animal models,...throughout the genome. However, their decreased sensitivity means that the best option moving forward may...most investigators use single cell clones for in vitro CRISPR experiments. The likelihood that a single...

...outdoors. Taking a walk or a bike ride is a great activity for both exercise and clearing your mind. And ...And because the outdoors is so huge, these are activities that you can easily do while staying away from... your life. At Addgene, we’ve started virtual activities like team lunches, a virtual soup lunch, and ...kids' needs. Plan ahead. Think about and set up activities that kids can do more independently. For example...meals and snacks ahead of time.  Find learning activities for kids. For example, Skype a Scientist is now...research community. Set expectations for your productivity Set a routine and stick to it. Plan defined...

...links the expression of gene III to base editor activity and editing speed and consists of the following...Figure 2: Phage-assisted evolution of base-editing activity. A plasmid-based genetic circuit in the bacteria...links the expression of gene III to base editor activity and editing speed. Created with BioRender.com....of TadA variants with much higher base editing activity compared to the original TadA-7.10 enzyme. After...showed increased off-target DNA and off-target RNA activity. To mitigate this drawback, the lab introduced...editor with improved Cas domain compatibility and activity. Nature Biotechnology. https://doi.org/10.1038...

...current understanding of neuroanatomy and neural connectivity, providing valuable insights for neuronal communication...methods have been developed to study neuronal connectivity. These methods are less neurotoxic and better...). When using these viruses to mark neuronal connectivity a large majority of neuronal cells are labeled...improvement resulted in a clearer picture of neuronal connectivity in the mammalian brain (Wickersham et al., 2007...tracing with AAV vectors is the Allen Mouse Brain Connectivity Atlas, a project to map neuronal connections... study can be found on the Allen Mouse Brain Connectivity Atlas website. A similar approach was used by...connections that are functionally relevant, i.e. activity-dependent neuronal tracing. Apart from mapping...

...off-target activity than BE3  85172  pBK-EQR-BE3  EQR-Cas9 (NGAG)  Lower off-target activity than BE3 ...donors in experiments simultaneously measuring the activities of CaMKII and RhoA during induction of structural...domain similarly allows for control of enzymatic activity. In addition, proteins can be “caged” by fusion...editing. For each base editor, they observed editing activity with a minimum efficiency of ~50% and confirmed...85177  pBK-YEE-BE3  SpCas9 (NGG)  Lower on-target activity than BE3; editing window ~1-2 nt  87438  pET42b-HF-BE3...pET42b-HF-BE3  HF-Cas9 (NGG)  Very low off-target activity; protein purification vector  87439  pCMV-HF-BE3...pCMV-HF-BE3  HF-Cas9 (NGG)  Very low off-target activity; mammalian expression vector Rees H, et al. Nature...

...recombinase activity were exposed to blue light. Cre-Vvd showed substantially improved activity as compared...permitting recombinase activity. Opto-Cre-Vvd is precise, demonstrating low sensitivity to ambient light, ...gene expression tools in and of themselves, their activity can be fine-tuned and precisely controlled through...authors demonstrate that CRISPR-Casɸ is active in vitro as well as in human and plant cells. Casɸ...

...for in vitro cleavage, but they improve cutting efficiency and are important for robust activity in cells... helps limit off-target activity. What next? It’s not actually clear! In vitro, Cas9 holds tightly to ...cells (Jiang & Doudna, 2017). Within an interior cavity, Cas9 grips nucleotides 1-10 of the guide RNA, ...the HNH domain also allosterically controls RuvC activity through a hinge region between the two (Jiang ...finish:   Figure 9: Cartoon summary of Cas9 activity. The apo enzyme is shown in the open conformation...

...voltage sensitivity of the available VSDs has been successful even at the single neuron level in vitro. In...function depend on potential energy. Cellular activities like action potentials, muscle contractions, ...effective at the single cell level as they are in vitro. Factors like photobleaching, lack of red-shifted...genetically encoded component is delivered in vivo or in vitro and a small molecule is added exogenously to measure...red fluorescent voltage sensor to detect neural activity. Sci Rep 9, 15878 (2019). doi: 10.1038/s41598-...

... Overview of in vitro AAV CRISPR screens. Created with BioRender.com. The in vitro option requires... screens may be AAVantageous!       In vivo, in vitro, ex vivo CRISPR screens can be carried out using...using a few different methods — in vivo, in vitro, and ex vivo. The general process of these screens is the...with transducing cells with the CRISPR library in vitro before transplanting them into mice. For in vivo...having an efficient and effective delivery method is vital for the best outcomes. The go-to in vivo option ...

...characterization of two Cpf1 orthologs that display cleavage activity in mammalian cells. Like Cas9 nucleases, Cpf1 ...see if any Cpf1 nucleases would exhibit in vivo activity in mammalian cells. From 16 diverse Cpf1 candidates... et al. found two that display robust cleavage activity similar to that of Cas9. These two nucleases, ...-base sequence. Double mismatches ablated Cpf1 activity, except when they were present in the 3’ end of... as Kleinstiver et al. saw no decrease in Cpf1 activity with 4-6 base deletions at the 3’ end of the target...found that both orthologs show lower off-target activity than that previously observed with SpCas9. Kleinstiver...

... website, write a blog Pursue speaking invitations by inviting others (be on a speaker committee) Follow...on the UK Career Site for Researchers called Vitae. Vitae recently published a career development framework...are always excellent Technical knowledge and creativity Become a “thought leader,” develop a technical...

...APEX suffered from inconsistent activity levels and limited sensitivity under the stringently low expression... of biogenesis and mitochondrial degradation in vitro and in vivo. Merged green and red channel fluorescence...that Opto-RTKs have a similar level of background activity and activation as their wild-type counterparts...proximity-dependent proteomic mapping with far greater sensitivity at lower expression levels. APEX2 is a monomeric...system Interested in optimizing TALEN assembly and activity? The laboratory of Takashi Yamamoto has created... of both the TALE scaffold and module on TALEN activity in a single-strand annealing (SSA) assay. This...non-RVD variations”, were found to improve TALEN activity. Each positional group of module plasmids (ex....

...magnetically sensitive and could manipulate cell activity in vitro. The Guler lab went on to test Magneto2.0 ...(Q3) | December 2016 (Q3) MARCH 2016Enhancer activity mapping with STARR-seq   Although many technologies... identification and quantification of enhancer activity have remained challenging due to the variable ...genome-editing tools that are deployed in both in vitro cell systems and diverse model organisms. Addgene...verified in zebrafish and was shown to offer high activity and unparalleled specificity in genomic targeting...cells using the Protein A tag. The luciferase activities can then be measured and normalized to assess...interactions. Higher relative firefly luciferase activity in the Co-IP compared of a control Co-IP indicates...

...recombinases used must have high activity and specificity with low cross-reactivity, as well as low toxicity....efficiency of only 70%. To determine the cross-reactivity of these recombinases, each recombinase and reporter...excision efficiency. B2 and R showed partial cross-reactivity; however, the other recombinase/reporter pairs...proliferating Drosophila cells by estrogen-dependent activity regulation. Heidmann D, Lehner CF. Dev Genes Evol...

...encodes endonuclease and reverse transcriptase activities.    Figure 1: Overview of retrotransposon...improves on SB100X by having 30% higher transposition activity (Voigt et al., 2016). Sleeping Beauty plasmids...mobilize DNA over 100 kB in size and is active in vitro as well as in vivo in yeast, plant, insect, and ...transcription start sites, CpG islands, and DNaseI hypersensitivity sites. Like Sleeping Beauty, piggyBac has ...transcription start sites, CpG islands, and DNaseI hypersensitivity sites. Tol2 is only active in vertebrates ...Tol2 and has ~3-fold increase in transposition activity  Tol2 transposon plasmids    Applications of...

...targeted cells makes it possible to control neuronal activity with a specificity far greater than the one that...the axon’s membrane, thereby controlling neural activity (Bernstein and Boyden, 2011; Mei and Zhang, 2012...light activates halorhodopsins to silence neuronal activity (Figure 1) (Wiegert et al., 2017). How to get...specific wavelengths, selectively turning neuron activity on or off (Atasoy et al., 2008; Taylor et al.,... this question, mapping and characterizing the activity of the DA neurons of the VTA. Using in vivo optogenetic...genetically targeted optical control of neural activity." Nature neuroscience 8.9 (2005): 1263. PubMed...