...Cas9 orthologs to identify a more efficient, well tolerated, Cas enzyme. They identified the Cas9 orthologue... essential genes in a wide variety of bacteria including in pooled, genome-wide, formats. Interested in...dCas9 variant as well. Furthermore, they provide a high-fidelity (HF) variant of the BE3 that further improves... Then, pCRISPR is introduced along with an oligonucleotide repair template. Through recombineering, the...in Streptomyces  Streptomyces bacteria produce a wide variety of bioactive natural products. To easily...INTEGRATE (Insertion of Transposable Elements by Guide RNA-Assisted Targeting) and the Zhang lab's system...select genes in E. coli. Furthermore, to demonstrate wider applicability they demonstrated success of Cas9 ...

...our CRISPR guide for a refresher on CRISPR/Cas9.  Figure 1: Cpf1 is a single RNA-guided endonuclease...crRNA/tracrRNA hybrid guides needed for Cas9 function. Since both Cpf1 and its guide RNAs are smaller than...Cpf1 functions differently. Since Cpf1 loci are widely distributed across bacterial species, Zetsche et... crRNAs for Cpf1-mediated cleavage are 42-44 nucleotides in length, about the same size as Cas9’s, but... cleaves in a staggered fashion, creating a 5 nucleotide 5’ overhang 18-23 bases away from the PAM. With...SpCas9. Since Cpf1 doesn’t require a tracrRNA, crRNA guides are only ~42 nt long. Direct synthesis of these...and off-target efficiency When Cpf1 was first identified, we didn't know much about its on-target and ...

...a march in support of science, I loved the idea and decided to go. At my local March for Science in Albany...advocate for scientist participation in government, evidence to inform policies, a protected place to work,...hired hinges on performing near miracles. When evidence does not affirm our perception of how things are...scientists are tempted to seek out alternative evidence and discard inconvenient findings. Yet, we cannot...scientists to rally in Ottawa to protest the “death of evidence,” and to speak out in unprecedented force during... and actions beyond marches, as well as allies outside the scientific community. Vanessa Sung is currently...Centre at McGill University in Montreal, Canada.  Outside the lab, she dabbles in science policy, public ...

...DNA edits and to perform functional screens to identify genes involved in a phenotype of interest. This...discuss differences between these approaches, and provide updates on how best to design gRNAs. You can also...gRNAs! Comic by Maya Kostman.   Important considerations before you start an experiment with CRISPR ...expression. Location and sequence are important considerations for designing your gRNAs. For indels, it's ...off targets. For CRISPRa and CRISPRi, these considerations are of roughly equal importance (target should...since potential target sites occur ~1 in every 8 nucleotides, restricting gRNAs to 5 – 65% of the protein ...with a PAM preference of NGG, is still the most widely-used Cas enzyme, the development of SaCas9, NmeCas9...

...programmable guide sequence as the spacer and the remaining sequence as the scaffold. gRNA: "Guide RNA" is ...between the guide and target in order to cut DNA, whereas some mismatches can be tolerated outside this region...CRISPR array. The CRISPR array is composed of identical repeat sequences and variable spacer sequences...includes the crRNA and tracrRNA sequences in a “single-guide RNA” that performs both functions. Cas9 cuts both...all have in common is the ability to use an RNA guide to target other nucleic acids—usually DNA—to cut... sequences in the CRISPR array that will become guides for future targeted degradation. Before integration...foreign DNA that Cas proteins recognize using an RNA guide. Non-target strand: The paired strand of foreign...

...do you identify the actively silenced ones? Here, I’ll cover some screening methods to identify them. ...RNAi-trigger type you decide, any RNAi experiment benefits from an easy method to identify and quantify actively... example, while gene knockouts have long been considered the “gold-standard” for LOF studies, the high...eukaryotic process where approximately 20-30 nucleotides of double-stranded RNA (dsRNA) results in downregulation...the importance of using orthogonal methods to confidently conclude the function of a gene of interest. ...RNAi activation. A typical RNAi experiment can be divided into three fundamental steps: creation of the ...Consequently, I will focus on different approaches and considerations present at the first stage of an RNAi-based...

...with Peptide-Assisted Genome Editing (PAGE). They fused the Cas enzyme to cell-penetrating peptides and...gene delivery capsids previously identified in rodents have not been widely successful in primates. The authors...articles.  Here's what you'll find in this post: Peptide-assisted genome editing for primary cells Monomeric...delivery Recombinant anti-FLAG tag antibodies Peptide-assisted genome editing for primary cells By Susanna...and co-incubate it with an “assist peptide” that facilitates endosomal escape. Combining Cas9-PAGE with...with a 30-minute incubation, even with multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused...reporter (mChe), with co-incubation with assist peptide (AP). C) Workflow for Cas-RNP-PAGE editing of primary...

...putting these into slide format. You want people to focus on you and your ideas. Slides will be     distracting...also be a slide deck, markers, etc. Chalk or markers force people to be more succinct. Slide decks leads...is vague. Instead, consider "A mechanistic understanding of this system will provide the first defined ...avoid the temptation to provide too many details. Reverse engineer your talk. Identify the key points you ...original blog post below! --Ed.  Eight years ago, I decided to write a "how to" manual on applying for faculty...the trials and tribulations of the process, I’ve provided guidance and mentoring to several people that ...failure to clearly articulate one's talents and ideas. Some key points from my ebook, "Applying for a ...

...programs provide support for academic career development, it's often left to students to identify and build... design variables need consideration? Enrich your knowledge and skills outside of the lab Effective verbal...you build a network outside of the academic world Participating in activities outside of the lab expands...and government. This great resource allows the undecided to learn about a variety of science professions...on their skills and interests. Once scientists identify potential career pathways, education and mastering...mastering new skill sets must occur by doing work outside of the lab. Extracurricular activities don't have...tool and technique development. However, careers outside of the lab highlight “transferable” skills such...

...specificity is dictated by a DNA guide sequence rather than an RNA guide as with Cas9. Third, it can be ...with an ssDNA guide. Therefore, researchers must co-transfect cells with 5’-P-ssDNA guides and an NgAgo...Cas9/gRNA complexes or with guide RNA- and Cas9-expression plasmids. Cas9 guide RNA delivery does however...sharing the stage with much-lauded CRISPR-Cas9 is DNA-guided endonuclease NgAgo! We'll discuss how NgAgo is ...have their own pros and cons (2). The NgAgo DNA guide is inexpensive and can be 5’-phosphorylated in house...modified from a company. However, unlike an RNA guide, it cannot be produced from a plasmid in cells. ...Try NgAgo for Yourself On the other hand, the widespread success of CRISPR-Cas9 across multiple model ...

...Yiqun, et al. "CRISPR Cas9-guided chromatin immunoprecipitation identifies miR483 as an epigenetic modulator...system, consisting of dCas9 and targeting gRNA, to identify molecular interactions at this site (Fujita and...Subsequent mass spectroscopy studies allowed for the identification of proteins associated with the IRF-1 locus...different fields of study. In addition to the identification of DNA binding proteins, this method can characterize...sensitive and specific and allowed the Xu lab to identify protein and nucleic acid binding partners at numerous...various biological fields. In 2015, the Fujii Lab identified non-coding RNAs associated with telomeres by ... malignancies. Additionally, the Tapscott Lab identified regulators of DUX4, a transcriptional regulator...

...skills guide Resources on Addgene.org Learn more about Careers at Addgene Watch out career videos  ...training. Similarly, there are several factors to consider when selecting a postdoc mentor and lab. These...Depending on the position, you may also want some evidence of teaching experience. How do you get there... having sufficient data to write a manuscript. Consider questions like: What figures are missing? For ...explore your system more, you will have a better idea of how to leverage it to ask new and interesting... grad school, you searched for and eventually identified a postdoctoral mentor. A good mentor typically...typically writes the grants that fund your research, guides the research directions of the lab, gives you constructive...

...to a target polypeptide. Multimerization in the SunTag system occurs via antibody-peptide labeling; specifically...they righlty deserve, but, in this post, we'll provide a small sampling of the many amazing new plasmid...-2xNLS-VP64 and dCas9(N)-FRB-NES). This system provides users greater temporal control over CRISPR/Cas9...successful in regulating gene transcription using DNA-guided optogenetic tools; however, targeting the necessary...overcome many of the limitations intrinsic to DNA-guided systems such as TAL effectors or Zinc Finger Nucleases...Nucleases, the Gersbach lab has modified the RNA-guided CRISPR-Cas9 system to create a tool that is quick...flexibility of the LACE technology makes this system widely accessible for many potential applications. Polstein...

...go wrong. We hope that we have provided you with a few ideas to consider if you do run into a roadblock... faced with a roadblock. In this post, we will provide an overview of some of the common challenges associated...such events, newer lentiviral packaging systems divide the viral elements among multiple plasmids. Second...packaging systems please see Addgene’s Lentiviral Guide. While the third generation packaging systems offer...experiments a bit more challenging. With these considerations in mind, when planning an experiment users ...transgene, and downstream applications should all be considered when choosing the correct system. Recently, ...transfer plasmids with small inserts (such as CRISPR guides) while a single 72h harvest is best for larger ...

...digest, PPIs are identified via mass spec analysis. Biotinylated proteins are considered possible interaction...distinct functions. Many existing methods for identifying PPIs, such as affinity purification mass spectrometry...spectrometry (AP-MS), lack the ability to specifically identify proteins that interact with a particular protein...the validation of a binary PPI as well as the identification of additional interacting factors. Problems...detect weak and/or transient PPIs. Many PPI identification techniques rely on protein interactors to stay... AP-MS. This requirement often prevents the identification of weak and/or transient interacting proteins...potential interactors with a bait protein but the flip side is that it’s difficult to know which proteins interact...

...microscopy techniques   Fluorescent Widefield Microscopy Fluorescent Widefield Microscopy is the most common...to solve two drawbacks of widefield and confocal microscopy. First, widefield and confocal microscopy project... visual system relies primarily on contrast to identify individual objects and perceive the world around...When performed correctly, fluorescence microscopy provides a high contrast image in which a bright signal...contrast, color contrast, to an image and thereby provide molecular or structural specificity to the viewer...microscope objective evenly illuminating the entire (wide) field of view. Fluorescent light traveling back...one time, limiting photobleaching. Similar to widefield, the entire field of view is excited at one time...

...Here, we will provide an overview of how to create and use a standard curve and provide some general considerations... considerations for scientists planning to use them in their assays.  What is a standard curve? Scientists...serial dilution of a control protein, incubated alongside an unknown sample with the BCA reagent. The absorbance... a known control sample, called a standard. The ideal standard curve has at least five dilutions, with... the data points generated by replicate values provides useful information about the accuracy of the curve... must be included every time the assay is run, ideally with freshly prepared buffer. This is because standard...users aim for an R2 above 0.95.    Additional considerations When running assays that rely on a standard...

...newer lab members. You must get an idea of what to expect and decide if it is what is best for you. Some...development? To get a better idea of whether or not a lab head is a good role model, consider questions like: How...publication record show evidence of industry collaboration? If you are considering a career at a small, ...focused on choosing a lab, but almost all of these guidelines can apply to any workplace or job. It amazes ...sections below. Mentoring relationships can help identify your priorities and needs, but sometimes it just...talk to as many lab alumni as you can or schedule video/calls if they are not local. Alumni meetings can...Former lab members can give you a fuller picture. Consider talking to people in the lab across the hall or...

...experiment - there are a number of outside factors that need to be considered as well. In this post, we’ll ...overnight, so mark the vial, add the incident to your records, and consider the following questions as you ...ll touch on antibody storage, buffer considerations, and give just the lightest of nods towards conjugates... and illustrious lab career, you will likely accidentally leave your antibody out on the bench overnight...1: Blugene fading into darkness because they accidentally left their antibodies out. It's okay, Blugene...could affect the reaction. For instance, sodium azide, a common antimicrobial agent added to buffers, ...reaction. Many commercial antibodies come with sodium azide already included in the buffer, which will be noted...

...common, besides being one of the five senses? GPCRs or G-protein-coupled receptors (GPCRs)! Aside from these...molecule ligands such as clozapine-N-oxide (CNO). DREADDs are ideal for chemogenetics as they are relatively...quick guide to chemogenetics Resources on Addgene.org Check out Addgene's chemogenetics guide Find chemogenetics...with heterotrimeric G proteins on the intracellular side of the cell membrane. Heterotrimeric G proteins ...Gα), beta (Gβ), and gamma (Gγ). G proteins are divided into four subtypes, based on their Gα subunit: ...that activate many GPCRs are still unknown. To identify some of these unknown ligands and their GPCR binding...Roth lab showed that this platform accurately identified 120 known GPCR targets demonstrating its potential...