...they think of these particles as tiny DNA cutting ninjas that deliver CRISPR to multiple types of targets...fluorophore or tag. To utilize HDR, a donor template containing the desired sequence flank by homology arms must...hydroxyphenylpyruvate dioxygenase (HPD) gene, all nine treated mice had ~10% editing in their livers, while...line on Nanoblades Nanoblades are tiny DNA-cutting ninjas which can stealthily delivery many types of CRISPR...

...and base changes from A•T-to-G•C are mediated by adenine base editors (ABEs). How does this work? Through...dilution (passage from the infected to a fresh uninfected host cell culture). This dilution step represents...passaged from an infected host-cell culture to an uninfected culture which results in less overall dilution...BioRender.com. Introducing ABE8e - a fast and flexible adenine base editor Lower-stringency PANCE, followed by...efficient in on-target DNA editing as ABE8e while maintaining the lower off-target DNA and off-target RNA editing...pathogenic point mutations. Together, cytosine and adenine base editors can correct all four of these kinds..., Liu DR (2020) Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility...

...to enable both negative and positive selection screening. They and their colleagues including Feng Zhang... (PubMed). Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem O, Sanjana NE, Hartenian...Dec 12. (PubMed). Genome-Wide Recessive Genetic Screening in Mammalian Cells with a Lentiviral CRISPR-Guide...

...an international researcher who uses plasmids containing genes from rabies and vesicular stomatitis virus...

...limited - what cloning approach should you use if the budget is particularly tight? Most cloning processes ...for traditional restriction cloning. Sequence and Ligation Independent Cloning (SLIC) could also be a great... The general Gateway cloning protocol is fairly flexible and adaptable to cloning different types of DNA...guarantees, some cloning techniques do tip the scales in your favor. For starters, Golden Gate cloning is extremely...since CRISPR; you are ready to clone! But...how? Cloning is no longer limited to restriction enzymes - although...: Speed Cost Difficulty Efficiency For cloning in particular, another important consideration ...inserts ...as some methods are more amenable to combining multiple DNA fragments than others. In today’s...

...advance their research on diseases of glycosylation, arginine mutagenesis, and neural development. Paula Videira...inherited metabolic diseases with severe and life-threatening consequences. They result from defects in different...drugs can be used to treat these diseases (drug repositioning). The ultimate goal of this work is to lower...PubMed PMID: 27393411. Michael McMurray: Rescuing arginine mutations in rare disease with guanidine Michael... mutations that change protein sequence affect arginine (Arg) amino acids more frequently than any other...

...some of them containing fluorescent markers or protein tags, and suitable for molecular cloning. Plus, we...bacteria and prep the plasmid before you begin your cloning experiment? What if after receiving the plasmid...collection has been prepared as part of our new cloning grade DNA service and is ready to use out of the...the box. We estimate that providing cloning grade DNA could save scientists up to three days of work. ...discovery.” Here are the details. Plasmids offered as cloning grade DNA As we begin testing out this service,...directly used in the research on the SARS-CoV-2. Nine of the overall plasmid selection will be open to...of target organisms (from bacteria to mammals), cloning techniques, and experimental use (CRISPR, viral...

...Adenine base editors (ABE) mediate A•T-to-G•C base changes (Figure 1), but it can be challenging to make...et al., 2020. Directed evolution identifies adenine base editors with improved editing in cell lines...TadA sequences to create the eighth generation of adenine base editors, called ABE8 (Gaudelli et al., 2020...Cas9 proteins to increase the targeting scope of adenine base editors The most commonly used S. pyogenes...(“N” can be any nucleotide base, followed by 2 guanines). This limits the use of ABEs as DNA sites that...off-target RNA and gRNA-dependent DNA editing while maintaining on-target base editing functionality.  The delivery... LE, Ciaramella G (2020) Directed evolution of adenine base editors with increased activity and therapeutic...

...Iterative improvement - Making Binning Singletons better Our experience running this program at ASM Microbe...This post was contributed by Joe James from Binning Singletons. The sheer scale of a large conference...bench scientist working at an institution where training opportunities weren’t always available. Even more...Denise Akob and Geof Hannigan that the outline for Binning Singletons came together. The term Singleton was...any reason was a Singleton. Figure 1: The Binning Singletons logo. Image: James et al., 2020. ...similar sequences together in bins, we thought that binning Singletons together would provide a structure and...Singletons in their bins.  The next component of Binning Singletons is when we bring all the bins together...

...circular polymerase extension cloning) and SLiCE (Seamless Ligation Cloning Extract) to name two more. Likewise... Assembly (NEB) Cold Fusion Cloning (System Biosciences) In-fusion Cloning (Clontech) Although these ...smaller fragments. Know your cloning methods There are two other common cloning methods that are easy to ...different methods. Golden Gate cloning does result in the seamless joining of fragments, but uses site specific...your insert. Gateway cloning utilizes λ integrase to catalyze directional cloning of DNA parts that are..., Gateway, or Golden Gate cloning methods. Whether it is for routine cloning, assembly of multiple fragments...About Other Cloning Techniques Additional Resources on Addgene.org Find Plasmid Cloning Protocols Find...

...encoded fluorescent sensor for serotonin. According to Unger, “serotonin is a well-known but poorly studied...medium-throughput screening, she increased the affinity of the sensor for serotonin by over four orders...leads to the release of serotonin (measured using the newly developed green serotonin sensor). In preliminary...use the serotonin sensor in sleep-related studies and in assays for measuring human serotonin transporter...Chiasson explains. Elizabeth Unger: Developing a serotonin sensor The goal of Elizabeth Unger’s postdoctoral...there are no known bacterial proteins that bind serotonin. Unger redesigned the binding pocket of the periplasmic...periplasmic binding protein so that it would recognize serotonin. Using a combination of computer modeling, directed...

...2in1 vector cloning system Article contributed by Michelle Cronin  Listen to the 2in1 cloning podcast segment...traditional Gateway™ cloning, the GOI first needs to be cloned into an entry vector. The 2in1 cloning system contains...restriction digest cloning to introduce the GOIs into the 2in1 vector instead of the GatewayTM cloning BP reaction...Recently, the Grefen lab introduced the “2in1” vector cloning system - a Gateway compatible approach to simultaneously...the GOI occurs through recombination. The 2in1 cloning system is well suited for protein-protein interaction...coexpression. By introducing a single plasmid containing both GOIs, expression levels are more evenly ...controlled. The Grefen Lab has utilized the 2in1 cloning system to generate a series of plasmids to study...

... Figure 1: Initial steps in the CRISPR-pooled screening using sgRNAiBAR. Image from Zhu et al., 2019.  ... Peking University has developed a new CRISPR screening method that makes use of guide RNAs with internal... After infecting cells with sgRNAiBAR library containing lentiviral vectors the iBARs can be detected ... annotated human gene, it enables genome-wide screening with a high MOI, which is beneficial for a low... calcineurin dynamics and can serve as a drug-screening platform to identify new antifungals that target... assay, and are not suited to high throughput screening, calcineurin dynamics measurements, or monitoring... that this method is compatible with dead cell staining using propidium iodine (PI), allowing for calcineurin...

...purpose: fast growth, high-throughput cloning, routine cloning, cloning unstable DNA, preparing unmethylated...Streptomycin MC1061 derivative. General cloning and storage, blue/white screening, leucine auxotroph. F- endA1 recA1... DH5alpha   General cloning and storage of common plasmids, blue/white screening. F- endA1 glnV44 thi...glnV44 λ- JM109   General cloning and plasmid maintenance, blue/white screening, partly restriction-deficient...NEB Stable   For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences...JM109-derived. For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences...from HB101. For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences...

...Direct staining is quicker than indirect staining and eliminates potential non-specific staining that may...can use both direct and indirect staining methods. In direct staining, the primary antibody against a ...intracellular staining where binding of large immune complexes may be hindered. Indirect staining can be beneficial...also keep their targets in mind when planning fixation. If staining both cell surface and intracellular...control is critical for determining the background level of non-specific staining.  If you’re using antibodies...-specific staining associated with the primary antibody. When following an indirect staining protocol,...omitted from the staining protocol. This control allows you to detect background staining arising from the...

...students, rotation students, joining a new lab at any career stage, learning a new technique while visiting...participant in the training. In this blog we will review the preparation and planning you can do to build...true for training in the lab! If you don’t do proper preparation and pay attention during training, then ...questions to your training! All of these things will help you get more out of the in-person training component...need to double-check a detail! Training follow up After your training session (ideally in the next few... the information in your training. Bench mentor-mentee relationships Training can be a very fulfilling...visiting a lab, or maybe even training on a new technique with a coworker. Being a new trainee (even if you...

...constructs? A new cloning system combining the advantages of Golden Gate and Multisite Gateway cloning methods ...that you need. The Golden GATEway cloning kit simplifies the cloning process for complex DNA constructs...vectors via traditional restriction enzyme cloning, TA cloning, or annealing of oligonucleotides. These ...efficient and flexible cloning process provides improvements to classical cloning methods, particularly... MoClo modular cloning system Synthetic biologists have developed a modular cloning strategy, MoClo,...assembly steps. Before beginning, scientists can insert fragments of DNA containing basic parts (promoters... of interest are retained after SLIC cloning. The general cloning strategy described in the associated...

...building block for further chaining. The MXS-chaining kit contains 5 empty chaining vectors plus a destination... 19; 22(2):169-74. High efficiency cloning for enzyme screening Dr. David Ackerley’s lab at the University...expression containing a variety of N- and C- terminal tags. The Santalucía Lab has developed a cloning toolkit...Multisite Gateway cloning. A desired insert is cloned using traditional restriction enzyme cloning to create ...(5):493-8. MXS cloning platform for imaging and flow cytometry applications Designing customized fluorescent...Neveu laboratory designed a new iterative, chaining-based cloning method that simplifies the process of constructing...with MXS-chaining. Analogous to modular assembly methods, such as BioBricks, the MXS-chaining method uses...

...any well-executed cloning endeavor, NEBuilder requires upfront planning. This cloning methodology requires...? In the context of cloning, DNA assembly refers to a method of physically joining multiple fragments ... the joining reaction. BioBrick Assembly will leave scar regions at the site of fragment joining, but ... for seamless joining of multiple mutations spread over thousands of base pairs. Cloning sgRNAs Generating... More about Gateway Cloning Gibson Assembly and Other Long-Homology Based Cloning Methods Screen Plasmids...end of a fragment. This means that fragments (containing regions of homology) cut from plasmids via restriction...the regions of homology. A powerful resource for planning an NEBuilder HiFi DNA Assembly mix reaction is...

...process of cloning, combining, and testing these different parts can be quite arduous. The cloning process...of a good screening experiment can be found on our blog post entitled “Genome-wide Screening Using CRISPR...Moduclar Cloning (CIDAR MoClo) Parts Kit. This Kit consists of a variety of plasmids containing many different...compatible with both standard Gateway cloning and the FNLCR Combinatorial Cloning Platform (CCP); the latter of...terminators for easy cloning of genes and promoters. In addition to USER cloning, this toolkit is also...GPCRs in the human genome are orphan receptors meaning their ligands are unknown. Identifying new ligands...approach termed Parallel Receptor-ome Expression and Screening via Transcriptional Output-TANGO (PRESTO-TANGO...