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...activity in HEK293 by reducing editing from ~30% in control samples to 0-10% in Acr samples. Acr proteins can...When added to human cells six hours after the introduction of Cas9 RNPs, AcrIIA4 reduced off-target editing...specific site. 2. Temporal, spatial, or conditional control of CRISPR activity Regulating Acr proteins by inducible...molecules could achieve rapid, dynamic, and spatial control of CRISPR-Cas activity. For example, fusing AcrA4II...LOV2 domain allows anti-CRISPR activity to be controlled by blue light (Bubeck et al., 2018). 3. Reduce...Your Next Experiment Read more about Optogenetic Control of CRISPR   Resources on Addgene.org Find CRISPR...

...response (e.g., graft vs. host) as well as the introduction of the cancer-fighting component, CAR. Clinical... edit with the goal of ‘fixing’ mutations or introducing a desired edit, but this particular class of ...of treatments has quite the opposite aim: to destroy. One treatment currently in clinical trials includes...but are not limited to, ones targeting muscular dystrophy, cardiovascular disease, inflammatory diseases...by Kendall Morgan and updated in 2023 by Susanna Stroik.  References and Resources References Yuxuan Wu...blog CRISPR Antimicrobials Treating Muscular Dystrophy with CRISPR CRISPR 101: Drug Discovery and CRISPR...

... calcium binding proteins (e.g., calmodulin or troponin C). Their fluorescent readouts are used to monitor... is a FRET-based calcium reporter derived from troponin C. It is engineered with a reduced number of calcium...fluorescent state only by simultaneous (1) user-controlled illumination with violet light and (2) exposure...different versions optimized for neuron-specific, astrocyte-specific (GCaMP6 only) or mammalian cell expression...101: Viral Applications Viral Vectors 101: An Introduction to AAV Find AAV Calcium Biosensor Vectors at... Susanna Stroik...

...are monoclonal antibodies that are generated in vitro using synthetic genes that are typically expressed... a patient’s blood), scientists can use mass spectrometry to determine the amino acid sequences of the...process of antibody selection and maturation in vitro by selecting for antigen binding from libraries ...or mammalian display (Tsuruta et al., 2017). In vitro selection allows scientists to create antibodies...into an expression plasmid, the plasmid can be introduced into host cells, such as bacterial, yeast, or...on the Addgene blog Read our Antibodies 101: Introduction to Antibodies blog post Learn more about hybridomas...

...approach of CeNGEN, check out their publication.   3’UTRome The 3’UTRs of mRNA are important for post-transcriptional...expression in C.elegans. In this system a promoter controls the expression of a GAL-4 driver that can bind...also find additional SapTrap plasmids used for introducing point mutations that was published in a microPublication...Central PMCID: PMC3531189. Mangone, Marco, et al. "UTRome. org: a platform for 3′ UTR biology in C. elegans... L., & Jorgensen, E. M. "SapTrap vectors for introducing point mutations with unc- 119 + selection." MicroPublication...split intein for refined spatiotemporal transgene control in Caenorhabditis elegans." Proceedings of the ...

...placing FP timer DsRed-E5 under the control of Neurog3 (a gene controlling pancreatic differentiation) in ... (500 to 580 nm) over the course of 18 hours in vitro (see figure 1B in Terskikh et al.) and even displays...showed that their versatile timer could be used in vitro, in mammalian cells, in C. elegans, and in Xenopus...synthesized or long-lived transcription factors control gross changes in gene expression as new body sections...Pratik, et al. "A programmable synthetic lineage-control network that differentiates human IPSCs into glucose-sensitive...

...101 eBook! Researchers use various techniques to control protein expression for experimental, biotechnological...successful. Some mammalian systems also allow for control over when a protein is expressed through the use...Protein expression from each of these vectors is controlled by a different promoter resulting in different...to E. coli. Of all the vectors, pET, under the control of the T7 lac promoter and induced by lactose, ...researchers often place recombinant proteins under the control of the galactose inducible promoter (GAL). Other...cell-free expression systems, proteins are assembled in vitro using purified components of the transcription and...

...donor templates and sequencing primers for quality control. At such a scale, automation is crucial, both for... to avoid re-cutting when needed. It gives you control over your data. CrispyCrunch keeps a record of ...repair outcomes, which is important for quality control. The only ones we found that worked with NGS data... 0 and 100 for each guide, with 100 being the strongest interaction between the guide and the target and...interactions (e.g., scores <20) are reduced to 0 and strong interactions (e.g., scores >80) are transformed...weak gRNAs (CFD < 20) bind almost not at all, and strong gRNAs (CFD > 80) bind almost as much as perfect...

...enjoy seeing how our quality control process has changed with the introduction of next generation sequencing...sequencing results we analyze as part of our quality control process has steadily grown. On a busy week, we ... more than 200 plasmids as part of our quality control process. Consequently our team has refined our ...database sequence is ≥ 95%. [Our QC process would be trouble-free and much faster if 95% of our results were...Plasmids 101: An Inside Look at NGS Plasmid Quality Control Learn about our Snapgene-powered plasmid maps. ...

...members of the lab or those taking introductory lab courses. The Intro to the Lab Bench series is meant...supplement your teaching? You’re in luck! Addgene’s Intro to the Lab Bench series was born from the shift ...meant to be an informative introduction for those who are just starting their careers in the lab and even... need a refresher on some best practices for introductory science techniques. It’s also great for teachers...

...capsids (i.e. the part of the virus that determines tropism) have been developed that increase transduction...expressing plasmids is generated. Diversity is introduced into the cap plasmid library by inserting short...which, in the case of Chan et al, was neurons and astrocytes. Why screen for IV deliverable AAVs? Typically... AAV vectors expressing RGB proteins under the control of an inducer, i.e. tet-off transactivator (tTA...expression in the CNS and PNS Previously to broadly introduce a transgene into a specific cell type in the brain...fluorescent tag-then-express system that allows for controlled induction of multi-color labeling of neurons ...

...A few months ago, we shared an introduction to immunofluorescence (IF) - a common method for visualizing...of interest within a cell or tissue. In that introduction, we broke down the method into six general steps...improve antibody binding for some targets, they can destroy it for others. That said, organic solvents’ impact... K, Mareninov S, Diaz MFP, Yong WH (2018) An Introduction to Performing Immunofluorescence Staining. Methods...resources on the Addgene blog: Antibodies 101: Introduction to Immunofluorescence Antibodies 101: Epitope...Epitope Availability Antibodies 101: Introduction to Antibodies Resources on Addgene.org Find recombinant...

...researchers developed viral vector systems based on retroviruses like Moloney murine leukemia virus (MMLV). The...system that could do both, Addgene depositor Didier Trono and collaborators turned to the lentivirus HIV-1...plasmid contained a viral envelope to dictate the tropism, or types of cells the vector could infect. Finally...vectors are incredibly popular in the lab - the Trono lab’s lentiviral packaging and envelope plasmids...Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D. Science. 1996 Apr 12;272(5259):263-7. PubMed ...Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L. J Virol. 1998 Nov;72(11):8463-71. ...

...resistance is swift, as few as five years after introduction. Current antibiotic strategies are nonspecific.... Phage therapy fell out of fashion with the introduction of antibiotics, but the field is currently undergoing...antibiotic-resistant bacteria (Figure 2). The first phage introduced is lysogenic, integrating into the bacterial ...lytic phage-resistant. When the lytic phage is introduced, it targets only the bacteria that are antibiotic...gain resistance to a lytic phage through the introduction of a crRNA (shown in blue). Infection with the...Bacterial Genome Engineering Treating Muscular Dystrophy with CRISPR Gene Editing Using CRISPR to Edit ...

... it is essential to include proper controls. As a positive control, include a sample that is known to ...This method also requires more optimization and controls when developing, as secondary antibodies can also..., purified protein, or a peptide. The positive control will confirm that the procedure is working and ...and should always be included.  For a negative control, include a sample that does not contain the target...where the target is not expressed. The negative control will help you determine if there is non-specific...

...! The team used other molecular techniques to introduce time modulation of the TEV cleavage and improve...dCas-KRAB activator and the dCas-VPR repressor are introduced into the hPSC genome using AAV1 and contain an...Find the plasmids at Addgene! CRISPR RNP electroporation and AAV donor infection (CRISPR-READI) enables...new tool for synthetic biology uses CRISPRi to control gene expression. This includes a CRISPRi-based ...affinity), as well as GRAB_NEmut as a negative control. Find the sensors at Addgene! New yellow fluorescence...AAV5 and AAVrg. Find these AAVs at Addgene New controls, biosensor, chemogenetic and optogenetic AAV for...

...New compact, efficient prime editors By Susanna Stroik The Liu lab recently used phage-assisted continuous...limitations and enable impressive editing efficiencies in vitro and in vivo. Incorporating reverse transcriptase... vectors, CreVLPs, and eVLPs for in vivo and in vitro applications.   Figure 2: Modular features... enzyme, Lee and Cheah et al. achieved precise control over when and where the enzyme is active, drastically..., then the other, a dual-enrichment and mass spectrometry workflow identifies proteins bearing both labels...light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells. Nature Methods...

...unable to reach all regions of the CNS. PHP.B, a neurotropic variant of AAV9, is better than AAV9 at gaining...receptor used by PHP.B viruses. Creating PHP.B, a neurotropic AAV9 variant PHP.B’s specificity for C57BL/6J...CNS transduction and transduces the majority of astrocytes and neurons across multiple CNS regions. PHP....Ly6a seemed the most likely candidate due to its strong linkage to the PHP.B phenotype and its expression...mice strain specific Ly6a SNPs on PHP.B binding in vitro. They found that when LY6A contained C57BL/6J SNPs...PMID: 30819613. Hordeaux, Juliette, et al. "The neurotropic properties of AAV-PHP. B are limited to C57BL...

...size using electrophoresis, typically capillary electrophoresis. Capillary electrophoresis reads a fluorescent...process, see our blog post on NGS plasmid quality control. NGS is generally cheaper if you have many samples...to sequence our packaged viral DNA for quality control. This gives us a faster turnaround time than short-read...Validation: seqWell Addgene’s Tips for Plasmid Quality Control Additional Resources on addgene.org Molecular...

...most as cancer cells are amenable to culture in vitro. Virologists too have benefited from the ability... ‘Master’ stock, which can be stored in liquid nitrogen long term, preferably some of these vials will...will be stored in a separate backup liquid nitrogen store to prevent loss from equipment malfunctions. One...also monitor the stock levels you have in liquid nitrogen for your Working and Master stocks of each cell... laboratories to carry our independent quality control for a range of testing, from animal doping to forensics...Thermofisher Guidelines for Maintaining Cultured Cells Invitrogen Cell Culture Basics Guide Abcam Cell Culture ...