...HNH endonuclease domain. Cpf1 cleaves DNA in a staggered pattern and requires only one RNA rather than ...cleavage and G-rich PAM requirement. Zetsche et al. started from square one to characterize the Cpf1 nucleases...nt upstream of the PAM site, Cpf1 cleaves in a staggered fashion, creating a 5 nucleotide 5’ overhang 18...amino acids long respectively), both cleave in a staggered pattern similar to FnCpf1. Potential advantages... another option to the CRISPR toolbox. Cpf1’s staggered cleavage pattern opens up the possibility of directional...

...cytometry experiments can be a little challenging to understand if you’ve never done any flow. But in order to...to fluorescent markers and/or cellular dyes and stains. These will be used in a series of one- or two-....  Don’t be intimated if you’re struggling to understand how the researchers decided to place their gates...FACS plot and follow a gating strategy, you can start to ask why the researchers made the decisions they.../doi.org/10.5500/wjt.v8.i1.23 Jhunjhunwala, S., Aresta-Dasilva, S., Tang, K., Alvarez, D., Webber, M.,...

...production steps before you can start transducing your virus. Let’s start there!     Figure 1: ...services that can prepare the libraries for you.  Starting from plasmids requires amplification, verification...page with protocols boxed in red.    Unlike a standard single plasmid transformation, though, a pooled...disrupting plasmid representation. A good place to start is with a protocol for a library of similar size...

...destination vector carry different antibiotic resistance markers (indicated here by plasmid color), allowing...How to clone using Gateway technology To better understand the process, we’ll walk through an example experiment...expression elements (ribosome recognition sequences, start codon, stop codons, reading frame considerations...Gateway cloning kit, and MultiSite Gateway plasmids. Constant reading frame When you move a DNA fragment from...Gateway vector to another, the inserted DNA fragment stays in frame. High efficiency The positive (antibiotic...

...alleles in a variety of species, including the standard laboratory mouse. It’s also made targeted insertions...protein knock-in, suffer from low efficiency when a standard dsDNA repair template is used. The original design...Easi-CRISPR efficiency. Easi-CRISPR is compatible with standard microinjection and electroporation methods using...editing components to the germline. In contrast to standard microinjection, i-GONAD is performed directly ...Gurumurthy, Masato Ohtsuka, and Rolen M. Quadros for assistance with editing this blog post. References Yang,...

...these different transcriptional start sites in order to better understand isoform-specific gene regulation...each gene isoform has a distinct transcriptional start site. Because CRISPRa can be targeted anywhere in... Bdnf gene possesses 9 distinct transcriptional start sites, all of which yield the same BDNF protein....downstream of the TUJ1 coding sequence. ES cells that stably express the dCas9-Suntag activator were transduced...

...size. From the pool of shorter orthologs, only Staphylococcus aureus Cas9 (SaCas9, 1053 amino acids) displayed...genome modification, but they did so without a substantial immune response or toxicity. More AAV-based CRISPR...Harrington LB, Al-Shayeb B, Wagner A, Brötzmann J, Staahl BT, Taylor KL, Desmarais J, Nogales E, Doudna JA...Dohmae N, Ishitani R, Zhang F, Nureki O (2014) Crystal Structure of Cas9 in Complex with Guide RNA and...Zhang F (2015) In vivo genome editing using Staphylococcus aureus Cas9. Nature 520:186–191 . https://doi.org...

... time-consuming, and complex to use outside of standard laboratories. In the case of the COVID-19 pandemic...SHERLOCK to detect SARS-CoV-2 RNA. The test is started using RNA purified from patient samples and can... a different PAM site and generates 5’ and 3’ staggered ends after dsDNA breaks. The Doudna lab discovered...applications. The company Mammoth Biosciences has been started based on DETECTR technology with the mission to...s41587-020-0513-4 Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA (2018) CRISPR-...

...protein is up or downregulated, examine a protein’s stability or post-translational modifications, or study ...A and Protein G, which bind to the fragment crystallizable region (Fc) of antibodies with a high affinity...conformation. Frequently, the antibodies used for a standard, denaturing Western blot will not be suitable ...interest, such as a sample transiently transfected or stably infected with the protein of interest and a sample..., Hjelm K, Sjöquist J (1972) Protein a from Staphylococcus aureus. Its isolation by affinity chromatography...

...controls for flaws. Recently the Bassik Lab at Stanford developed a new genome-wide CRISPR knockout screen...via a lentiviral vector to cell lines that either stably expressed Cas9 or that had been lentiviral infected...also lead to fewer false-positives results (i.e. mistakenly calling a gene a hit when really it’s not). In...the PAM are less tolerated than mismatches more distal to the PAM, and 2) guides with high GC content ...error rates in genomic perturbation screens: gold standards for human functional genomics." Molecular systems...

...longer shelf life and the development of disease-resistant crops to increase agricultural yield (Wang et ... experiment. CRISPR components can be expressed stably or transiently depending on the delivery method...be delivered and expressed transiently using a standard detergent, Polyethylene Glycol (PEG), although...Y, Liu Y-G, Zhao K (2016) Enhanced Rice Blast Resistance by CRISPR/Cas9-Targeted Mutagenesis of the ERF...homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol 32:947–951 ...

...be used with Type IIS restriction enzymes and standardized primers to clone your gene of interest into ...clone your gene of interest. Translation of the standardized leader peptide prevents secondary structures...The gene set was carefully chosen to perform statistically relevant benchmarking of cell-free expression... daptomycin reveals an ordered progression to resistance." Antimicrobial agents and chemotherapy 57.11...effects during adaptive evolution of antibiotic resistance." Proceedings of the National Academy of Sciences...

...videos for you to level up in the lab, and we’ve started collecting some of our favorite (moving) images... You’ll be able to use these GIFs on Twitter, Instagram, TikTok and more. Just type “addgene” into the...looking for that perfect GIF for your TikTok or Instagram story, we also have stickers to help express your...check out everything we’ve uploaded so far, and start sharing the science love today!        ...

...promoters. Others are only active under specific circumstances. In a previous post, we discussed inducible ...which can be switched from a default OFF to an ON state, and how you might use these in your research. Today...which can be switched from a default ON to an OFF state, as well as repressible binary systems. In recent...GAL4 gene downstream of a tissue- or developmental stage-specific promoter and design a UAS reporter construct...QUAS promoter is less leaky than UAS in the basal state, and co-expression of the inducer QF can increase...

... critical systems that cannot be removed or substantially reduced in an attempt to save energy. The transition...renewable electricity is certainly not the only sustainability effort we’ve implemented at Addgene. The kitchen...check out Leila Haery’s blog series, “Bringing Sustainable Practices to the Lab.”  Whether participating...blog Subscribe to the Addgene blog Read our sustainability in the lab blog posts Read another blog post...

...transfection in: 15 cm dishes OR CF5 stacks Select how many dishes or stacks you plan to transfect: 1 to ...following μg of each plasmid needed per dish or stack. Calculations are based on using 28 μg of total ...) per 15 cm dish or 2500 μg of plasmids per CF5 stack. Total ug of each plasmid needed for the whole experiment...

...million bacterial stabs take up? If 81 vials fit into one freezer box, one million stabs would take up over... community (check out the infographic below for stats on our plasmid deposits and requests). Over the ...needs of the scientific community. For example, we started a new service distributing ready-to-use viral preps...

...We often think of DNA as inert. It generally stays put, which makes it easy to locate in a genome. But... in bacteria, transposons can move antibiotic resistance genes between plasmids or from plasmids to the...piggyBac is biased to integrate at transcription start sites, CpG islands, and DNaseI hypersensitivity ...Tol2 also prefers to integrate at transcription start sites, CpG islands, and DNaseI hypersensitivity ... of RNA guided transposons in bacteria sets the stage for combining the super powers of CRISPR and transposons...

...both brain function and dysfunction. First, let’s start with the basics: What is optogenetics? Optogenetics...Controlling neurons with light In 2005, a team from Stanford University, led by neurobiologist Karl Deisseroth... central nervous system due to their relatively stable extra-chromosomally long-term expression and low... arousal in mice previously subjected to steady-state general anesthesia (Taylor et al., 2016). These ...therefore, an ideal partner to optogenetics to understand the cellular and molecular mechanisms of the ...

...education content. This work exemplifies our long-standing commitment to be responsive to the community’s...environment. In particular, we are working to understand what challenges remain in materials sharing, ...innovation around the world. As always, we seek to understand our role in opening up science to help create...therefore embarking on a strategic refresh to understand this new world and our place in it. This period...