...BioRender.com. The pegRNA specifies both the genomic location to be edited and the sequence of the edit, so ...its design changes for every new prime editing application. And optimizing its sequence is important, as...left), while pegRNAs that use other bases in this location bind to Cas9 with canonical sgRNA structure (right...structured motifs like mpknot, use the pegRNA Linker Identification Tool (pegLIT) to create linkers that minimize...

...most obvious example of an individual part, this category also includes promoters, UTRs, terminators, etc...multi-purpose it can be. Let’s review a list of MoClo applications. Genetic circuits Most things in biology are...performing production and subsequent necessary modification of these chemical products. The flexible nature...genotype and phenotype is best described with “it’s complicated.” Applying MoClo Interested in using MoClo? You...

... therapeutic potential in mice with a dominant cataract disorder caused by a single copy of a gene known...known as Crygc (Wu et al). They found that the cataracts phenotype could be rescued by zygote co-injection...substantial relief from both disorders. As of publication, submissions to the UK, EU, and USA federal drug...10.1186/s12943-022-01559-z Zarghamian P, Klermund J, Cathomen T. Clinical genome editing to treat sickle cell...

...sufficient spectral overlap, the fluorophores must be located within 1-10nm of each other and be oriented appropriately...decision easier. 1. Müller, Sara M., et al. "Quantification of Förster resonance energy transfer by monitoring...have enhanced the ability of labs to answer sophisticated questions about cellular processes. Currently...PMC18018. 2. Miyawaki, Atsushi, et al. "Fluorescent indicators for Ca2+ based on green fluorescent proteins ...

...prevent the formation of replication competent lentivirus (RCL). Replication competence can arise from...end-users, size of the transgene, and downstream applications should all be considered when choosing the correct... as resources available, cost and downstream applications. For example, while the PEG precipitation method...

...there are two main decisions to make: 1. Which modification? and 2. Which viral vector should be used? Modifying...excitation or inhibition upon light (or drug) application.  Genetic manipulation can also reduce expression...recombinase on transgenic flox animals to introduce modifications at the DNA level in a temporally and locally... brain area, avoiding side effects from such modification in other areas. An alternative strategy is to...

...a method that uses loop-mediated isothermal amplification after reverse transcription (RT-LAMP) and a ...receptor for SARS-CoV. The lab also found that cathepsins play a role in SARS-CoV infection and deposited...deposited plasmids for the expression of cathepsin S, L, and B. This became a great starting point for SARS-CoV..., Zengel JR, Sathyan V, Nguyenla X, Van Dis E, Catamura C, Yamashiro LH,  Begeman A, Stark JC, Shon DJ...

... labeling proteins with different subcellular locations. Image from Goudeau et al., 2020. Goudeau... show a high proportion of blue fluorescence, indicating cell proliferation. BaF3 cells maintained in ...in high IL-3 showed mostly blue fluorescence, indicating a quick cell cycle. Cells that had IL-3 withdrawn...Hybrid for Multiplexed Editing and screening Applications (CHyMErA) Knockout Libraries express hybrid ...

..., 2018). For example, AcrIIA4 mentioned above indicates that it’s the fourth Acr protein discovered that...viral particles that have the gene deletion could replicate when challenged with the S. islandicus CRISPR-...B, Bondy-Denomy J (2020) Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies....light-inducible Cas9 activity? Check out these these plasmids Catch Up on Your CRISPR Background with Our Guide Pages...

...some new and popular split-FP plasmids from our catalog to help you find the right split-FP plasmid for...pairs with N173 derived from Cerulean or Venus Identification of new fluorescent protein fragments for bimolecular...fluorescent proteins for protein labeling and signal amplification. Zhou S, Feng S, Brown D, Huang B. PLoS One....Huang Yellow Venus pBiFC-VN173, pBiFC-VC155 Identification of new fluorescent protein fragments for bimolecular...

...approach (spoiler alert: it’s dCas9… with no modifications). Ready to get started?   Figure 1:... when DNMT1 methylates the genome during DNA replication, it doesn’t methylate sites blocked by dCas9....for dCas9 to interfere with DNMT1 even through replication. The dCas9 also needs to be removeable, otherwise...vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat Biotechnol 34, 1060–1065. ...

...genome editing technology that allows precise modifications of DNA (replacements, small insertions, and ...configurations can be used to introduce specific target modifications. Since this means we could design hundreds to...affects prime editing outcomes across many genomic locations. In our study, we used the TRIP (Thousands of ...to chromatin data from over a thousand genomic locations. This allowed us to train ePRIDICT (Figure 3),...

...methodologies, with a special emphasis on these five categories. Please note that many of the following cloning...relies on site-specific recombination reactions catalyzed by BP Clonase and LR Clonase enzyme mixes. After...there is no need for restriction digests, gel purifications, or ligations. The general Gateway cloning protocol...Chaokun, et al. "FastCloning: a highly simplified, purification-free, sequence-and ligation-independent PCR ...

...but will also give you a chance to slow down and catch your breath before diving in to the next figure....don’t fully understand the question, ask for clarification. Having them reframe or elaborate their thought...system, and is interested in a career in science communication.       Resources on the Addgene blog Read other... other posts on scientific communication Find more tips for scientific networking Read this blog post ...

...The vast majority of bacteria are undomesticated which limits the tools scientists can use to study them...the genome and is propagated during normal DNA replication and cell division. Mobile-CRISPRi does not disrupt...single-gene knockdown experiments. Mobile-CRISPRi applications Mobile-CRISPRi expands CRISPRi to non-model ...Elizabeth A., and Pamela A. Silver. "Harnessing undomesticated life." Nature microbiology 4.2 (2019): 212....

...Starting with libraries and a biotinylated (for purification purposes) protein target of interest, the protocol...-30) of sybody candidates are sequenced for identification. All unique sybodies can be subcloned into ...2020) Generation of synthetic nanobodies against delicate proteins. Nature Protocols 15:1707–1741. https...Light to Manipulate Cell Signaling and Protein Purification RANbodies: Reporter Nanobody Fusions Plasmids...

... sequencing are more definitive, but visual identification is a good place to start before taking on more...resources are limited or for bioart and teaching applications. Find a chromoprotein for your experiment! ...events: The plasmid carrying the desired genetic modification integrates into the genome (Figure 3). Here,...of Allelic-Exchange Vectors for the Scarless Modification of Proteobacterial Genomes. Applied and Environmental...

...upon growth on LB/Cam/X-gal/IPTG agar plates, indicating successful reconstruction of the coding sequence... sequence-dependent ligation efficiency and identification of mismatch-prone pairings. Using these 4 bp...Kucera is a Principal Development Scientist of Applications & Product Development focusing on DNA cloning...Optimization of Golden Gate assembly through application of ligation sequence-dependent fidelity and bias...

... millions of people die from cancer-related complications each year. Despite these grim statistics, new...any of these genes through several different applications such as fluorescent protein fusion studies and...Lemieux is a Scientist at Addgene with interests in education and scientific writing.     References 1. Martz...Martz, Colin A., et al. "Systematic identification of signaling pathways with potential to confer anticancer...

...strategies is strongly advised. The effects are multiplicative, and there are very few gene drive experiments...An accidental release could delay real-world applications against scourges such as malaria and dengue ...about gene drives can be found in the referenced publications. Thank you to our guest blogger! An inventor...2003). PubMed. Esvelt, K. M., Smidler, A. L., Catteruccia, F. & Church, G. M. Concerning RNA-guided gene...