...candidate in the Sternberg Lab at Columbia University Medical Center. DNA transposons are ubiquitous genetic ...a multi-spacer CRISPR array. Workflow and technical considerations Making insertions in bacterial ...into cells and plate overnight Screen colonies phenotypically or by PCR/sequencing While CRISPR-transposons...few important details. For transposons to be specifically recognized during transposition, genetic cargos...Additionally, these transposon also exhibit something called target immunity, whereby multiple proximal insertions...Leveraging a unique marriage between distinct biological elements, CRISPR-transposons excel at programmable...candidate in the Sternberg Lab at Columbia University Medical Center. He currently studies CRISPR biology and...

...purification (TAP) once referred specifically to a combo tag comprised of a calmodulin binding peptide (CBP), a...to) purification, detection, solubilization, localization, or protease protection. Thus far Plasmids 101...overexpress and purify proteins can face any number of technical challenges depending on their protein of interest...consider junk protein. Though there are a number of chemical and peptide-based proteosome inhibitors, glutathione... relatively small sequence of amino acids, is basically a molecular leash for your protein. If you’re ...want to combine a fluorophore with a tag that localizes your protein to the nucleus? Or you want multiple...

...Harvard Medical School, and is currently a fellow in clinical genetics at the University of California, Los...Vincent and Niroshi Senaratne, from the Biological and Biomedical Sciences program at Harvard University...the Program in Genetics and Genomics at Harvard Medical School. He studies developmental gene regulation...

...Emily Bentley Building on their CROPseq method for optical pooled CRISPR screening, Paul Blainey’s lab has...multiplexing approach to Cas9-based pooled CRISPR screens called CROPseq-multi (Walton et al. 2024). This platform...compatible with enrichment, single-cell sequencing, and optical pooled screens in human cells. Addgene offers vectors...one of the virus’s most abundant proteins and a critical component for viral genome packaging. Addgene ...Genetic modification of microglial immune cells is critical for central nervous system research; however, ...cells.      Figure 5: A) Representative confocal images showing GFP expression from ssAAV5s with...gene of interest, reducing the immunogenicity typically associated with viral vector-based therapies. ...

...results (i.e. mistakenly calling a gene a hit when really it’s not). In practical terms, this means that... approach for identifying genes involved in a biological process. These screens provide an alternative...cutting than full-length guides (19-20bp)?  Small-scale studies (Tsai et al., Fu et al.) suggest 17-19 bp...non-targeting guides on screen analysis and hit calling. 5,644 non-targeting guides and 6,750 safe-targeting...ricin screen. When safe-targeting guides are used to call hits from the growth screen, p-values were less ...References 1. Morgens, David W., et al. "Genome-scale measurement of off-target activity using Cas9 toxicity...

...Harvard Medical School, and is currently a fellow in clinical genetics at the University of California, Los...Niroshi Senaratne and Ben Vincent from the Biological and Biomedical Sciences program at Harvard University...the Program in Genetics and Genomics at Harvard Medical School. He studies developmental gene regulation...

...provided here for reference: OpenWetWare Preparing chemically competent cells (protocol)  Preparing electrocompetent...protocol)  Untergasser Lab Preparation of chemically competent cells (protocol) Preparation of electrocompetent...until we are ready to perform transformations. A typical 200mL bacterial culture produces 20mL of competent...transformation protocols below: OpenWetWare Transforming chemically competent cells (protocol)  Electroporation (...protocol)  Untergasser Lab Transformation of chemically competent cells (protocol)  Transformation of...plasmid is introduced to bacteria, the bacteria is typically grown in liquid culture without antibiotics to...

...able to bind to a small molecule derivative of GFP called 3,5-difluoro-4-hydroxybenzylidene imidazolinone...intracellular locations using Cas9 and track the localization of the complex. Broccoli, the other green fluorescent...Broccoli, and useful for quantitative studies. Specifically, Corn was developed to quantify RNA transcription...high-affinity complex for RNA labeling and tracking." ACS chemical biology 9.10 (2014): 2412-2420. PubMed PMID: 25101481...and directed evolution." Journal of the American Chemical Society 136.46 (2014): 16299-16308. PubMed PMID...di-GMP and cyclic AMP-GMP." Journal of the American Chemical Society 135.13 (2013): 4906-4909. PubMed PMID:...a photostable RNA–fluorophore complex." Nature chemical biology 13.11 (2017): 1187. PubMed PMID: 28945233...

...populations; for example, CD8+ T (“T killer”) cells. Typically, you would label the CD8 co-receptor protein on...of interest via FACS using negative selection is called dump gating. Let’s say we want to sort out all ...cell membrane but inside the cell. Due to their chemical nature, antibodies cannot penetrate the cell membrane...intracellular labeling with antibodies requires chemically pre-treating the cells to allow for entry of ...esterified dyes that can enter cells freely are typically used. Inside the cell, the ester group is cleaved...intracellular labeling protocol is necessary, requiring chemical treatment of the cells after surface labeling....Associate in the lab of Arlene Sharpe at Harvard Medical School. His projects include investigating negative...

...colleges, private universities, and medical schools. I will emphatically deny that I have a magic formula..." manual on applying for faculty positions in biomedical science. My motivation was to share my experiences...told all of the nuances of the research in as technical of language as possible. Seriously. My suggested... trying to say X." She would then look at me quizzically and ask why I didn't just say that? Bottom line...especially helpful. I set out to make something practical and accessible. If there's something you come ...the Janelia Research Campus of the Howard Hughes Medical Institute. His interests include the quality control...

... why you might want to be careful relying on so called “housekeeping proteins.” Fully quantifying a western... later steps in this chain (blue) to infer or calculate things about earlier ones. In practice, multiple...popular choices for loading controls. These are often called “housekeeping proteins” and are detected with an... will almost certainly be overloaded. One paper called this problem “the most common error associated ...Data is presented as mean ± SEM. Adapted from Analytical Biochemistry, vol 440, Gilda, J. E., & Gomes,...loading control to β-actin for Western blots. Analytical Biochemistry, 440(2), 186–188. https://doi.org...Antibodies 101: The Basics of Western Blotting Technical Design of a Western Blot Troubleshooting and Optimizing...

...where you can start to identify which items aren’t critical to your research success. If you can get away ...resources to keep, and which ones to abandon. It’s critical to make these decisions in the following order... budget can help you compare the monetary and empirical value that helps you determine whether or not ...know how to decide which components are the most critical to your research, negotiations can be much easier...negotiating finances, instead of solely discussing numerical values, try to keep conversations focused on research...saving the institute time and energy that would typically be wasted going through some of the questions ...is your goal, then understanding your needs is critical to a successful negotiation and collaboration....

...you’ve saved. Repurposing regulated medical waste Regulated medical waste (RMV), which we collect in those...scores products by their environmental impact on a scale of 1-10: the lower the score, the lower the environmental...companies to obtain these labels! Reusing plastic Historically, scientists washed and reused materials such...flow cytometers. These fluidic machines handle biological samples and are cleaned between each use. You...those red biohazard bags, typically is destined for a landfill. However, Triumvirate Environmental developed...

...deaminase (TadA) to a catalytically impaired Streptococcus pyogenes Cas9. They called this editor Target-...-to-inosine (A-to-I) RNA editor called REPAIR by fusing catalytically inactivated RNA-targeting Cas13 ...plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools more ...editor still retains A-to-I editing. This system is called RNA Editing for Specific C-to-U Exchange, or RESCUE...

...temporal and spatial control. Optogenetics uses genetically encoded tools, such as microbial opsins, to control...light-inducible heterodimerizing cryptochrome 2 (CRY2) and calcium and integrin-binding protein 1 (CIB1) proteins....this approach. This study generated the first optically active saCas9 protein. Stop light! Photoactive...different approach. These researchers used a genetically encoded photocaging technique to insert a light-removable...light-removable protecting group, specifically a nitrobenzyl photocaged lysine (PCK), on the Cas9 protein...Hemphill et al. presented data showing that this genetically encoded, photocaged Cas9 system could silence...editing and transcription. Label genomic loci Photochemical Control Hemphill et al., 2015   Nitrobenzyl-caged...

...Regulatory DNA function is typically inferred based on correlative biochemical characteristics and ectopic...Canver of Boston Children’s Hospital and Harvard Medical School. Addgene is proud to present a video reprint...for deletion screening by conventional PCR. Biallelically disrupted cells can be easily identified. Even...investigator at Boston Children’s Hospital and Harvard Medical School. His lab studies the molecular genetics ...Matthew Canver is an MD/PhD student at Harvard Medical School in the laboratories of Stuart Orkin and ...

...Kristin Krukenberg and Jessica Polka from Harvard Medical School and a long list of supporters from all of...peer review and grant awards systems need to be radically overhauled.  Finally, the science publishing system...excellence to begin effecting change.  Panelists call for action Each of the speakers in the panel had...Director of the Office of Postdoctoral Affairs at UNC, called her presentation “Disequilibrium, Disillusion and...Associate Professor of Systems Biology at Harvard Medical School, titled her presentation “How to Train Your...and take action. More Reading: “Rescuing US biomedical research from its systemic flaws” by Bruce Alberts...

... then study where the protein is localized, and how its localization and expression may change across ...transcriptional activation. Multiple nuclear localization signals (NLS) increase protein import into the...technology, the Vale and Weissman labs took this biological lesson and created a system to amplify fluorescent...modified the GCN4 sequence to increase its alpha-helical structure and stability, creating v4 SunTag. Since... also affect protein activity, half-life, or localization, these concerns are greater with SunTag. v1 ...

...poster tubes (or as our baffled flight attendant called them, "long, skinny things") and generally exuding... tales of back country skiing, fly fishing, and local grizzly bear attacks. He described one such recent...All Addgene depositors! Addgene was mentioned specifically in Dr. Zhang's introduction. His willingness...Cas9 that is improving the nuclease specificity dramatically and reducing off-target effects. Other young...discussed some of the many challenges of creating biological circuits, and I can only say this science is ... deposited and distributed – have changed so dramatically in the past 4 years. I kept busy for the whole...

...expression by various mechanisms. The RNA guide is typically 20 nts and the genomic locus must often be proximal...promiscuous. In other words, they can sometimes localize to unintended sites with sequence similarity to... the on-target site. When a CRISPR-Cas system localizes to unintended sites and performs its programmed...product with CRISPR to (hopefully) be used in human clinical trials. An off-target rate of 5% is far too high...Cas-OFFinder, CCTop, and many more. While such software typically only considers DNA sequence and doesn’t take into...deadCas activator/repressor experiments where only localization of the Cas occurs. 2 gRNA approach Off-targets...effectiveness as a control. Since off-targets typically confound an experiment due to their location (...