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...and primary cells. References 1. Doench, John G., et al. "Rational design of highly active sgRNAs for CRISPR-Cas9...These libraries utilize optimized sgRNAs that were designed based on the rules identified in Doench et al....1,000 non-coding control sgRNAs. This library was designed to improve on-target activity while reducing off-target...with 6 sgRNAS, highlighting the benefit of sgRNA design in CRISPR libraries. Thus, the Brunello library... that if an essential gene plays a role in the desired screening phenotype, it may be missed because it's... the sgRNA metrics, confirming again that sgRNA design plays a significant role in library efficiency....CRISPRa, sgRNAs were targeted between 150-75 nucleotides upstream of the TSS. Similar to the Brunello ...

...testing. You can find Nick on LinkedIn. References 1. Skloot, Rebecca, and Bahni Turpin. The immortal life...contamination and match their original description. The oldest, largest, and most diverse of these repositories...culture in the present day Cell culture is a widespread tool used in the fields of oncology, virology...problems surrounding cell culture over the last few decades some pharmacologists have tended to avoid cell ...International Cell Line Authentication Committee includes a database listing all cross contaminated or misidentified...comparable studies to be performed on a cell line even decades after it is initially isolated. Regrettably this...Master and Working stocks to work from. The future Attitudes are changing but yet more is needed to convince...

...plasmids are passed down through generations (Figure 1). These so called “plasmid addiction systems” require...than plasmids without this DNA. They eventually designated this fragment as the ccdB region (for “coupled... serves as a counterselectable marker in the destination vector. Successful cloning occurs when the ccdB...restriction endonucleases in plasmid addition was first described by studies of EcoRI and Bsp6I in Escherichia coli...systems. As with the other toxin-antitoxin systems described above, one part of the pair is more stable than...toxin-antitoxin system discovered. Unlike the systems just described, the hok/sok systems consist of three components...long-term experiments. They’ve also helped researchers design and optimize cloning methodologies using counterselection...

...drives our bioinformatics needs       Fig. 1: QC'ing data at Addgene is like standing under a Niagara...of data. In this two-part blog series, we will describe the importance of bioinformatics at Addgene and...each week. Our internal software was originally designed to help us work in large formats, such as 96-well...Thanks to the flexibility inherent in Nextflow’s design, we were free to use multiple programming languages...alone! The benefits of containers are they are designed to ‘contain,’ in a complete package, all of the... who or which computer ran the process. Another design choice we appreciated about Nextflow was how effortless...small with a print statement and tested that the desired data was available so we felt confident we were...

... done and we’re onto the next story. Figure 1: Addgene’s Agile Approach Sharing is caring There...released a major redesign of how search works on our site. For Addgenies, development includes everything from...experience and the overall soundness of the software design. Each of us brings slightly different cognitive...Addgene. Another advantage of Scrum is that it provides transparency to everyone involved about the status... on a new feature. We start with a story that describes the feature and what the criteria is for accepting...our users, we are able to make informed software design choices, resulting in software that helps our users...

...inclusion bodies and is folding properly. References 1. Angov, Evelina, et al. "Heterologous protein expression...features to consider when designing a full gene. A non-exhaustive list includes: Codon abundance relative...that their codons are more appropriate for the desired expression host. This is known as “codon optimization...as easy as it sounds. Even for relatively short peptides, there can be many possible ways to encode them...every amino acid I’d like to encode,” but, as described above, not every region of a protein should necessarily...PubMed Central PMCID: PMC4794256. This review provides a great overview of codon usage bias 8. Tuller...

... end sequences (R and L ends).  Figure 1: (A) Components involved in each type of CRISPR-Transposon...human therapeutics (e.g. using Sleeping Beauty). Despite their diverse utility, DNA transposons have been...(V. cholerae INTEGRATE - hereafter VchINT) and encodes a Type I-F CRISPR-Cas system. The four major components...the same promoter, and a donor plasmid (pDonor) encodes the mini-transposon. pEffector and pDonor can also...: Clone spacer(s) into the CRISPR array Clone desired DNA cargo into the mini-transposon Transform plasmid...even very large genetic cargo, at one or multiple desired genomic loci. Many applications of these systems...

...mammalian expression from human elongation factor 1 alpha Constitutive  Tends to give consistent expression...insert into RNA. In practice, the term "promoter" describes the combination of the promoter (RNA polymerase...   Primarily used for RNA     transcript     Description Expression Additional considerations CMV General... phosphoglycerate kinase gene.  Constitutive  Widespread expression, but may vary by cell type. Tends ...promoters     Promoter     Primarily used for Description Expression Additional considerations T7 in ...identify the right tissue-specific promoters as described by researchers at the NIH here.  Note: A. Max...

... reached labs in over 75 countries. Figure 1: SARS-CoV-2 proteins and other components. Image from... developed based on 100 structure-guided spike designs and has improved protein yields and stability. ...Plasmids deposited from Alejandro Balazs’s lab includes those for generating pseudovirus and those encoding...Liu Y, Chou C-W, Byrne PO, Hjorth CK, Johnson NV, Ludes-Meyers J, Nguyen AW, Park J, Wang N, Amengor D, ...Finkelstein IJ, McLellan JS (2020) Structure-based design of prefusion-stabilized SARS-CoV-2 spikes. Science...

... of low-toxicity RNA editing tools.   Fig. 1 Schematic of luciferase RNA knockdown assays using...The hardest part of any revolutionary discovery, including CRISPR, is turning potential into impact. ...two pieces: a single protein, computationally described as a fusion of four Cas7 proteins and a putative...RNA editing methods — streamlining experimental design and its potential for clinical applications. Specifically...high-fidelity rate. Both Cas7-11 and PASTE open up multitudes of possibilities at the bench and potentially...

...processed since the declaration, only a small fraction (<1%) of authors have asked for advice about the scope...the time of manuscript submission. The rationale includes increasing the ability of reviewers (and, once...issue by requiring ever more condensed methods descriptions, legends and limiting numbers of figures such...could be mitigated by on-line services such as SlideShare, Figshare and Dataverse. Just as Addgene helps... raise the alpha isoform of GSK-3 to the same (deserved) stature as the beta isoform. Follow him on Twitter...

...can make this fun activity relate to STEM.” Figure 1 shows the activities we had for my school’s Math &... can teach  5-10 year olds in a single evening. Grades K-5 cover a broad range of attention spans, skill...CRISPR work? This section was essentially background slides getting everyone on the same page in terms of the... by running it by the teachers and sharing some slides on Twitter, where I was able to get some great ...

...two (Knutson et al., 2010a,b).      Fig. 1: Undergraduates in Provost’s CURE prepare to analyze...semester one and then proceed to use those skills to design and carry out their own novel experiments in semester...resources, offers mentoring for instructors, and provides opportunities for students to exchange ideas and...results in student learning gains and positive STEM attitudes in CUREs. CBE Life Sciences Education. 21(4):ar74...

...antibiotic selection. Essentially, this gene is a 2-in-1 providing both bacterial and eukaryotic selection,...The NPTII gene confers resistance to several aminoglycosides, including G418 which is commonly used for ...DNA and generating DNA breaks. The Sh ble gene provides zeocin resistance by expressing a protein that...experiments in which off-target mutations are undesirable. Pros: Works across many species/systems. Cons...

...antibody bonding strengths.    . Steps of an IP: 1) Protein extracts from cells or tissues contain a ...most situations.  A slightly different method is desirable if the antibody is expected to bind weakly to ...vary in shape and structure. Their porous surface provides a large area for binding, but antibodies conjugated... slightly higher, than those of agarose beads, despite the size difference. Magnetic beads are particularly...

...your virus. Let’s start there!     Figure 1: Flowchart showing the process for using pooled lentiviral... If not, our protocol on Lentivirus Production provides a general approach for producing lentivirus in...excellent protocol paper by the Feng Zhang lab describing construction, lentiviral production, transduction...the Broad Institute- A variety of protocols and guides regarding pooled libraries, from cloning a pooled...

...dollar contracts behind closed doors. Fig. 1: The mythical land of negotiations.    You negotiate...specific details about the individuals, each example provides a scenario where both people get something valuable...

...successful and meaningful career in the grants profession: 1. Build on your existing skills & passion  Skills such...LLC, a grant professional development firm that includes the social media community GrantChat. She has ... from administration and fundraising to program design and evaluation. She and her team at J. Miller &...

... such a popular model organism? Zebrafish (Figure 1) were initially recognized as a useful vertebrate ...deactivation method in zebrafish are short, modified oligonucleotides called morpholinos. When injected into zygotes...CRISPR/Cas9-mediated insertion of foreign DNA. Despite these caveats, several features of zebrafish—including...

...determine purity of the AAV preparation. Figure 1: Silver staining of purified and non-purified AAV ...protocols, titering methods, and quality control are described below. AAV Production AAV distributed by Addgene...) are generated from lentiviral preparations (described above) that are subject to a concentration step...