...E.coli, named TadA7.10 in ABE7.10)  a catalytically impaired Cas protein (dCas or Cas nickase)  a guide RNA...accessible. Image from Gaudelli et al., 2020. Directed evolution identifies adenine base editors with...editing in cell lines Nicole Gaudelli, the Associate Director and Head of Gene Editing Technology at Beam Therapeutics...insertions and deletions (indel) by the catalytically impaired D10A nickase from ABE7.10 can be a problem. Therefore...editors The most commonly used S. pyogenes Cas9 requires the target site in the genomic DNA to contain ...editors are precise molecular tools, they do have undesired off-target editing effects. ABEs have been described...off-target editing frequencies. For applications that require high DNA editing specificity, for example for potential...

...extra level of safety, they also require a fourth plasmid. The requirement for additional DNA can impact ...third generation systems such that it no longer requires the viral transactivator, Tat. For more information...increasing the number of recombination events that are required to generate RCL. To overcome the titering hurdles...unconcentrated prep. With this in mind, if the experiment requires a certain volume of virus, consider scaling up...larger vessel, such as a 3 or 5-layer flask will require some initial optimization; cell number, media volume... method is relatively inexpensive and does not require an ultracentrifuge, PEG will precipitate all proteins... may not be the best method when pure virus is required downstream. When assessing lentiviral titer, don't...

...tropism. Briefly, different envelope plasmids can direct the production of virus with various tropisms. ...therefore, reduces the number of plasmids that are required in the transfection step of the viral packaging...incompetent? Yes, as with lentiviruses, the genes required for γ-retroviral packaging and transduction are...

...expand applications that require very specific targeting, like homology directed repair and base editing...order to bind DNA, Cas9 and other CRISPR enzymes require a short PAM sequence adjacent to the targeted sequence...David Liu’s lab, have gone one step further, using directed evolution to create xCas9, an enzyme recognizing...more complicated if you’d like to use homology directed repair to make precise edits or knock-ins. Editing...other species, like S. aureus and N. meningitidis require different PAM sites than SpCas9, in this case, ...broader PAM flexibility. Creating xCas9 through directed evolution Hu et al. used phage-assisted evolution...about CRISPR Base Editing Learn about Homology Directed Repair Resources on Addgene.org Check out our...

...users to generate a plasmid containing a direct fusion of the desired gRNA to the U6 promoter, and a second...genomic site and, using the endogenous homology directed repair pathway, a new user-defined DNA sequence...designs either oligos or synthetic DNA for the desired gRNA target sequence, as well as the 5’ and 3’ ... and a Cas9 expression plasmid will insert the desired genetic tag and marker sequence into the targeted... the insertion, and 3) a vector containing the desired tag, which can be obtained as a universal donor...expression vector. They can also be introduced directly into a Drosophila S2 cell line stably expressing...homology to the gene of interest to promote homology directed repair after a CRISPR/Cas9 induced break. Learn...

...kind of resources - including your time - will be required to produce an outcome you’re happy with. Know ...semester-long after school program? Comparing your desired outcome to the limitations and potential of your... communications below!   Format Resource Requirements Pros Cons Video Medium to high Uses visuals.... Easy access from smartphones with low data requirements.  No visuals. Variety of hosting platforms can...distribution.  Static. Can be difficult to convey an entire story or context via still images. Some things ... Digital or print distribution.  Static. Often requires both artistic and scientific training. Cartoons...media type to another is cognitively demanding. Requires multiple areas of expertise.  Social Media ...

...quantity of DNA that is required and, as a consequence, also dictate the required sizes of the bacterial...extraction. Restriction enzyme based approaches can require several hundreds of nanograms or micrograms of ...Gibson assembly or Golden gate assembly use a more direct approach, allowing scientists to clone from a smaller...extraction. Figure 2: Use cloning grade DNA for direct cloning of your gene of interest into a plasmid... plasmid in mammalian cells and it will likely require micrograms of plasmid DNA for each reaction. As...chemical contaminants, these DNA samples must also be entirely endotoxin free. Endotoxin is a lipopolysaccharide...

... beginning of the coding sequence to delete the entire gene? Perhaps you are more interested in the c-...expression cassette. The target sgRNA can be cloned directionally into the BbsI site. (c) The pCAG-EGxxFP target...the two halves of EGFP to recombine by homology directed repair, and resulting in the expression of EGFP...component of the system. However, it is easier to directly inject the pX330 plasmids used in the validation...Working with plasmid DNA is much easier and doesn’t require the extra precautions needed when dealing with ...they are born, it’s time to screen them for your desired edit. Good news! Remember the primers you designed...genotyping), but bear in mind that this method will require you to house mice that may have no mutation, costing...

...don’t require the organism to be specified and are truly organism agnostic. Other tools require an organism...Multi-locus asseEENdb database ssment would be required to fully evaluate off-targeting, but currently...design. These databases are still young and will require a collaborative effort from the community in order... Advanced functions: These functions are not entirely necessary but should be considered extremely useful...available mostly work on any computer but generally require working knowledge of the command line or scripting...scripting. Input flexibility: Software tools require some kind of data input in order to generate results...it takes authors to add a genome to the tool’s repertoire. Organism agnostic tools are the better solution...

...doesn’t require a functional Cas9 nuclease, so there’s no potential for aberrant DNA cleavage. Direct HIV-... notably CD4+ T-cells, and eventually leads to acquired immunodeficiency syndrome (AIDS) in untreated ...infections not usually seen in healthy individuals. Antiretroviral therapy (ART) can virtually eliminate plasma... to survive, but preventing re-infection would require sustained expression of Cas9/gRNAs, which could...at the level of protein structure, CRISPR gRNAs require DNA sequence-specific binding. Patients’ HIV-1 ... and kill” strategy may be a better option than direct cleavage. Despite the potential difficulties in...Putatunda R, Young WB, Khalili K, Hu W. CRISPR/gRNA-directed synergistic activation mediator (SAM) induces ...

...primarily used for direct visualization while epitope tags are used for both direct and indirect visualization...must be performed after primary purification and requires a second clean up step to remove the cleaved tag...Also, purifying GST by affinity chromatography requires proper folding of the tag, which prevents insoluble.... Mammalian purification is still possible but requires optimization and often more stringent washing....especially if downstream antibody detection is desired… and yes, we have an antibody for that!   ...

...independent, it does not require purification of insert or vector products, nor does it require the use of specific...purification, and, most importantly, excessive time requirements. FastCloning is a simpler yet reliable cloning...Standard Restriction Cloning and Fast Cloning Requirement Restriction Cloning Fast Cloning Choose Restriction...templates. Digestion products are then transformed directly into chemically competent E. coli and transformants...competent E. coli for this procedure to work properly. Directly transform chemically competent cells with the ...Addgene Blog Plasmid Cloning by PCR Perform Site Directed Mutagenesis by PCR Catch up on Your Plasmid Background...

...materials. Problems with Chip-seq ChIP-seq typically requires a large amount of input material, cells or tissue...experiment can be a major technical hurdle.   ChIP-seq requires  cross-linking during an initial fixation step...sites when in fact they are just non-specific or indirectly interacting. The CUT&RUN method Enter CUT&RUN... the University of Washington. CUT&RUN doesn’t require fixation and significantly decreases background...wing as a model. A typical ChIP-Seq experiment requires around 1,000 wings. Collecting that much tissue...controlled by the addition of calcium, which MNase requires for its nuclease activity and is chelated from...cells or when a high degree of signal to noise is desired. It also provides an excellent additional measure...

...ensuring good communication. A manager must both hire and fire to make sure the team is the best it can be....organization and is also one of the most common criteria required for a promotion. Yet little explanation is given...quality. Their work involves coordination and can require technical mastery. Scientists need to be doers ...successful because you are not fulfilling the requirements of the current task. Most of us start as doers...may be happy with a primary role of doer for your entire career. Scientists train as doers and often maintain...download the Addgene Management for Scientists Ebook.  Fire, Snowball, Mask: How Leaders Spark and Sustain Change...

...bypasses the 5′ mRNA cap required for eukaryotic systems and instead directly engages the ribosome for... Good tools for measuring intracellular pH are required in order to determine the relationships between...fluorophore is pH-dependent. Signal is simultaneously acquired from the the fluorescent and luminescent siganls... a gene-specific forward primer containing the desired restriction site (the MCS has several options) ...specified order using Multisite Gateway cloning. A desired insert is cloned using traditional restriction ...vectors in a Multisite Gateway reaction with a desired destination vector expressing a separate reporter...MluI, XhoI or SalI sites must be removed by site-directed mutagenesis in order to use the module as a building...

...distinct proteins (Rep78, Rep68, Rep52, Rep40 - required for the AAV life cycle; VP1, VP2, VP3 - capsid...supplied in trans . In addition to Rep and Cap, AAV requires a helper plasmid containing genes from adenovirus...annealing, upon infection, which eliminates the requirement for host cell DNA synthesis. Unfortunately, this...

...just one injection step! The entire protocol takes about 2-3 weeks but requires less than eight hours worth...signaling a new era in C. elegans research. Homology directed repair (HDR), which enables insertion of custom... elegans/CRISPR experiments, but this strategy required the use of unhealthy, difficult-to-inject unc-...injected 60-80 animals per gene, they obtained the desired insertions on the first attempt for 6/7 genes. ...Dickinson et al. have developed a modular system that requires very little hands-on labor and could facilitate...Better Cas9 Editing Efficiency CRISPR 101: Homology Directed Repair  ...

...select first hires who are well-suited, productive, and fit well with your goals. These first hires will set...set the tone and atmosphere that future hires will look up to." She also recommends to seek out advice...hiring process. Read Genevive Bjorn's "Ready, Set, Hire" on Nature Jobs for some quick advice and resources...surprised by the variety of skill sets she needed to acquire as a professor. “Being a PI is more than just experiments...recognize that every graduate student or postdoc may require different types of mentoring. Some mentees prefer...lab and selectively picking the first couple of hires to build a strong, collaborative team. And take ...

...nucleotides within a defined region of a genome. Require stringent bacterial growth conditions. Expression...capable of mutagenesis in E. Coli, but this approach requires special bacterial growth conditions in order to...Phage Assisted Continuous Evolution (PACE) method requires constant turbidity of the culture, so bacteria...introduce mutations. However, by using nCas9 to direct PolI3m to a particular genomic loci, EvolvR allows...mutagenesis of a site of interest. A gRNA is used to direct the PolI3M-nCas9 complex to a DNA site of interest...PolI) that is fused to a nicking Cas9. A guide directs Cas9 to a site of interest, where it nicks its ...lambda red recombineering of bacteria Use site directed mutagenesis to generate specific mutations in ...

...Chromatin regulation is complex and influences--directly or indirectly--many other areas of biological research...looking for on this list, please consider searching directly on our website. In-exhaustive Chromatin-related... might be useful for your work. The links below direct you to pages containing lists of plasmids from ...publications. You can request all of these plasmids directly from Addgene. Note that there are plasmids from...recombination. Gong F, Chiu LY, Cox B, Aymard F, Clouaire T, Leung JW, Cammarata M, Perez M, Agarwal P, ...techniques, the posts below may steer you in the right direction! CAPTURE-ing Chromatin Interactions: Using CRISPR-dCas9...