...approximately 1,900 sgRNAs and identified sequence features and adjustments that improved sgRNA activity and...libraries (see below). Let’s take a look at the features and advantages of the optimized human CRISPR libraries...scientists to conduct genome-wide screens in standard cultured cells as well as more difficult conditions such...sgRNAs for CRISPR-Cas9–mediated gene inactivation." Nature biotechnology 32.12 (2014): 1262. PubMed PMID: ...and minimize off-target effects of CRISPR-Cas9." Nature biotechnology 34.2 (2016): 184. PubMed PMID: 26780180...activation by an engineered CRISPR-Cas9 complex." Nature517.7536 (2015): 583. PubMed PMID: 25494202. PubMed ...

...Chromatin folds into secondary structures (loops) and tertiary structures (globules) to achieve exquisite... enhancers with the help of genome math. Figure 1: Genome math illustrated. Intersect (left) and...which genes influence the survival of our cell culture is the foundation of modern drug discovery and ...periodically use next-generation sequencing to measure the relative proportion of guide RNAs (gRNAs) across...for further details. DNA is a 3D Fractal Figure 2: DNA compaction. Graphics courtesy of beyondthedish...

... without DNA cleavage. Nature 551:464–471 . https://doi.org/10.1038/nature24644 Glaser A, McColl B, Vadolas...APOBEC-Cas9n-UGI complex. So how exactly does this work? Figure 1: APOBEC- and Cas9-mediated editing (ACE) reporter...mCherry positive cells (Martin et al., 2019). Figure 2: Editing efficiencies using base editing reporters...visualized and sorted via flow cytometry. Figure 3: Targeting pEF-BFP with a cytidine deaminase ...detectable protein, not just fluorescent proteins. Figure 4: Reporters in Gene On include a mScarlet reporter...

...antisense RNAs inhibit plasmid replication.   Figure 1: The replication problem. Iterons are repeated...the same RepA molecules, forming a “handcuff” structure that physically blocks replication machinery when...actually does, and neither plasmid is maintained (Figure 1).  The partitioning problem Plasmid incompatibility...leading to plasmid loss (Diaz et al., 2015).  Figure 2: The partitioning problem. For low copy plasmids...type of plasmid into a separate daughter cells (Figure 2) (Ebersbach et al., 2005). However, more recent...Partition-associated incompatibility caused by random assortment of pure plasmid clusters. Molecular Microbiology 56:1430...

...discipline to solve them on their own. According to a Nature News Article, since the 1980s researchers have ... of all of this appears to be that video is the future in terms of communicating ideas. In 2012, for example...and a few other disciplines leading the way, the future of complementing published research with video ... helps researchers go beyond citation rates to measure the impact of their contribution. It offers a 5...pin/7439 Research Website: https://umaine.edu/aquaculture/ The goal of the WeShareScience project is to...Richard. "Interdisciplinary research by the numbers." Nature 525.7569 (2015): 306-307. 3. Spicer, Scott. "Exploring...

...From the beginning, one of Addgene’s goals was to ensure the scientific community obtained high-quality ... programs to a uniform language.  The modern architecture of Nextflow included built-in support for containers...convenient compatibility with our internal software infrastructure; however, it is not necessary to use containers...input channel to only use the first n items. To be sure, we went through much trial and error to learn the...with our bbduk process.    At first, to make sure we understood the concept of channels and implemented...compared to purchasing and maintaining our own infrastructure. Our patchwork collection of scripts evolved...

...experiments or even negatively impact cells (see Figure 1). When behaving properly, FPs enable researchers...inappropriate disulfide bonds in the secretory pathway (Figure 1) [4,6–8]. Additionally, FPs that encode N-glycosylation...from properly exiting the ER and fluorescing (see Figure 2) [5].  When using FPs containing two cysteine...experiments, please consider the following: How can one ensure that an FP fusion with a non-cytosolic protein ...cellular compartments including the secretory pathway (Figure 2), the inner membrane space of mitochondria and...proteins optimized for diverse cellular environments." Nature communications 6 (2015). Pubmed PMID: 26158227....

...bacteriophage lambda Can be temperature regulatable  Often paired with the temperature sensitive cI857 repressor...replication are necessary to replicate plasmids and make sure the cells maintains it. Here, we'll cover the promoter...transcribed from the RNAP III promoters. This post features promoters for general RNAP II and RNAP III transcription...retroviral constructs and we will discuss these in a future post on viral vector parts. Promoter specificity...type of RNA transcript, you will also need to make sure your plasmid has a promoter suited to working in...

...below: How-to videos Our How-To videos feature screen-capture footage to help you navigate Addgene’s ..., we have also created video protocols.  These feature step-by-step instructions to navigate various lab...biotech nonprofit? In our Career Video series, we feature interviews with real Addgenies who have a variety...Addgene.  You’ll hear all about their career paths, future goals, and get some useful career advice.  Our ...

...Make sure to check out our validated gRNAs to avoid rolling the dice on a new gRNA in the future! References...target cells with Cas9. Hooray! Now it’s time to make sure your genome edits went according to plan. In this...validation on your bulk population. This way you can ensure you aren’t looking for a needle in a haystack before...sgRNA-Cas9 combo. Pro tip: When designing your PCR, ensure there is at least ~200 base pairs of sequence flanking... inserted or deleted accordingly. Pro tip: make sure your PCR amplicon isn’t too large so that you can...

...biomedical research and drug development. Integrin structure and conformational complexity There are 24 known...With the right research reagents, researchers can measure this conformational change to investigate the role...specific integrins in cellular processes.   Figure 1: Integrins can shape into three conformations...much lower affinity than their open states (C). Figure courtesy of IPI. IPI anti-integrin toolset IPI...biology talks on the IPI website and stay informed on future events by signing up for the IPI newsletter. The...characterization of subtype-specific β1 integrin mechanics. Nature Communications, 13(1), 7471. https://doi.org/10.1038...

...applications.  Third, even in the most optimized tissue-culture based system, the efficiency of gene editing is...limitations, the AAV approach is best suited for tissue culture based approaches that require modification of only...mutations of a gene into the endogenous allele in cultured cells. AAV genome editing combined with targeted...T-cells and hematopoietic stem cells respectively. Future researchers could apply this technique to efficiently...Hirata. "Human gene targeting by viral vectors." Nature genetics 18.4 (1998): 325. PubMed PMID: 9537413...editing via postnatal administration of AAV vector cures haemophilia B mice." Scientific Reports 7.1 (2017...

...Scientists. Back when I used to do mammalian cell culture, I would often respond to friends asking me to ...achieving your goals. However, you should have a good mixture of useful tasks that can be accomplished quickly...Monitoring task progress. For this, I always make sure I have explicit goals in mind for the endpoints ...extensively, my lack of desire to go sit in the tissue culture hood may have prevented me from splitting cells...splitting cells boring, hanging out in the tissue culture room also gave me time to listen to some of my ...any other time management suggestions, please be sure to note them in the comments! Additional Resources...

...desired variants of a protein. Applying a selective pressure winnows the pool down to plasmids expressing variants...High-throughput DNA sequencing is then used to measure the frequency of each variant during the selection...plasmid will be degraded by ExoIII. See the below figure for an example like this. 2) When your gene of ...to remove the BbvCI site(s). Overall, the key features of the one-pot saturation mutagenesis method are..."Plasmid-based one-pot saturation mutagenesis." Nature Methods 13.11 (2016): 928-30. PubMed PMID: 27723752... a Large Scale Using Deep Mutational Scanning.” Nature Protocols 9.9 (2014): 2267–2284. PubMed PMID: 25167058...

...to recapitulate the true steric and charge-based nature of the phosphoryl-modification; they are both much...defined three components required for translation (Figure 1): A tRNA that recognizes a specific ‘open’ codon...encoding the open reading frame (ORF) of interest (Figure 2). 1. The phosphoserine OTS The backbone of the...be optimized for parameters such as induction temperature and length of induction, which will vary between...Philip. "The origins of protein phosphorylation." Nature cell biology4.5 (2002): E127-E130. PubMed PMID:... permits programmable protein phosphorylation." Nature communications 6 (2015). PubMed PMID: 26350500....

...editing efficiency. Read the paper in Nature Find the SECURE base editors All-in-one AAV delivery ...inhibition Covalent tethering of DNA template to Cas9 SECURE base editors Nme2Cas9 CRISPR interference in Candida...genome as a negative control. Read the paper in Nature Communications Find the E. coli genome-wide CRISPR...plasmids Selective curbing of unwanted RNA editing (SECURE) base editors Keith Joung’s lab recently found ...

...Based on CRISPR/Cas by Shravanti Suresh Guest blogger Shravanti Suresh who studies CRISPR at Iowa State...launched the cloning grade DNA service. This service features 210 plasmids: empty backbones, fluorescent protein...plasmid in TE, Tris, or water? And what about temperature? Find the details in this blog post. 9. Droplet...stably maintain them in the cell. This blog post features two mechanisms that cause plasmids to be incompatible...

...AacCas12b) exhibits optimal DNA cleavage at 48 °C, a temperature incompatible to genome editing in human cells...strand breaks at target sites. Read the paper in Nature Communications Find the Cas12b plasmids  Plant...AAV toolkit - this time using the Staphylococcus aureus Cas9 (SaCas9). This AAV-based genome editing system...highly customizable as vector backbones, and gRNA structure, numbers, and promoters can all be chosen. Read...transferred via conjugation.   Read the paper in Nature Microbiology Find the Mobile-CRISPRi plasmids...

...popular any one of them will become. One thing is for sure, as is obvious from our most requested plasmid, ...modified for viral trasduction (see the Zhang Lab's Nature Protocols Paper for more information). V2.0 This...included in this plasmid's name are some additional features. The first and most imporant is that, upstream...these variants, as well as links to the Zhang Lab's Nature Protocols Publication describing them here. References...Genome engineering using the CRISPR-Cas9 system." Nature protocols 8.11 (2013): 2281-2308. PubMed PMID: ...

...narrowly focused lectures, interactive panels, and a student poster session. All DayCon lecture content is ...variety of programs, including fall and spring lecture series, science cafes, and Reddit AMAs. Their website... engaged. The day kicked off with introductory lectures targeting three broad concepts: the genetic basis... what life is like as a student researcher. The future of DayCon DayCon and other similar events show ...