...not the case for knock-ins. Knock-in mutations typically require an exact DNA sequence to be incorporated...know that HR is often mutated in cancer cells, specifically in breast and ovarian lines commonly used in...common editing pathway for DNA repair. To tilt the scales in your favor for an HDR edit, you can try one ...

... familial aspect of Addgene. As our size has drastically increased, it would have been a tight squeeze...event! Addgene company culture At Addgene we organically developed the philosophy of  “work hard, play...department) right in your breakroom. 5. Throw some physical activity into the mix It’s easy to sit around ...

...community to help Involve family. If you don’t have local family to help, create “fake” family by forming ...help you do your job better. #8 Thou shalt enthusiastically foster independence Your partner can make ... fold their laundry, take public transportation, call a cab and remember their own school assignments,...

... relative to empty particles (black arrowhead). Scale bar = 100 nm.   AAV resources Browse AAV preps... a concentration step prior to being frozen. Specifically, lentiviral particles are collected from the... strain of E. coli. In addition, plasmids are typically prepared using endotoxin-free plasmid purification...

...are subject to excision and ssDNA cleavage by canonical base excision repair enzymes. At the same time...targeted by a cytidine deaminase base DNA editor. Specifically the BFP mutant (BFPH66) contains a histidine...markers, and other enzymes such as Cre-recombinase drastically expanding the base-editing reporter toolbox. ...

...by Joe James from Binning Singletons. The sheer scale of a large conference can be intimidating. And it...when we bring all the bins together at a mixer. We call this Quorum Sensing, after the phenomenon in bacteria... career scientists, students, and those from historically underrepresented groups. Through such mentoring...

...below. The GUS gene (β-glucuronidase) with histochemical GUS-staining is a very useful visual reporter...protein of interest to elucidate the subcellular localization of your protein of interest. Find all plasmids...see Figure 3). This method is probably the most scalable method. Transfer your plant into the dark 16 ...

...writing are key skills. Other fields, often highly technical or skills-based ones, may not use them very often...questions about your application. These could be logistical, like briefly explaining a gap in your job history...letter needs to be coherent and free of typos and grammatical errors. It does not need to be a literary masterpiece...

...generate a dot plot (Figure 1). Clustered events are called a population.                             ...of markers you use to define your population is called your “gating strategy.” This series of markers ...around clusters to define populations. This shape is called a ‘gate’; it tells the machine that everything ...

...SpCas9 and SaCas9, Zhang’s group used an approach called BLESS (direct in situ breaks labeling, enrichment...post-injection. Livers from these mice were histologically normal and liver injury markers were not increased...enough to deliver with AAV. This system fuses catalytically dead dCas13b to the catalytic domain of RNA ...

... D1135E variant is far more selective for the canonical S. pyogenes PAM sequence NGG compared to wild-...novel CRISPR proteins has and will continue to dramatically increase the number of CRISPR applications. ...Wolfe SA, Sontheimer EJ (2018) NmeCas9 is an intrinsically high-fidelity genome-editing platform. Genome...

...uses a “nicking” Cas9, in which one of Cas9’s two critical enzymatic residues has been converted to an alanine...deaminases edit A-to-G.) In short, the impaired Cas9 localizes the deaminase to where the edit is desired, and...to dCas9.  This technique has been exploited to localize factors such as transcriptional activators and...

... to synthesize a large volume of information: basically, a scientist’s tool kit. I wrote a review article... was cheating. Eventually I learned that it’s practical because there simply isn’t enough time to read...used both. Curate and present some useful data Typically lists and pictures are the most useful parts of...

...blocking off time for different tasks. I use google calendar for this and try to give myself flexibility by...activities that are needed to accomplish these goals. Practically speaking, this means I write everything in a ...down? It’s more viscerally appealing for me to physically cross things out (it just feels good) and I remember...

...even when larger vector genomes are packaged, the physical size of the genomes is still ~4.7kb. But despite...overlapping strategy has a region of homology, typically ~400-1400 bp long between part A and B of the ...overlapping split vector had an improvement in muscle physiological performance.  2. Trans-splicing Overview: For...

...clones from that population. You can additionally calculate the minimum number of clones you would have to...consider using a version of Cas9 that has been specifically engineered for reduced off target editing, such...Resources at Addgene: Read our CRISPRGuide Get practical help from our CRISPR Protocols Find gRNA Design...

...here). Cas9 is commonly tagged with a nuclear localization sequence to enhance targeting to the nucleus...interest (GOI) into plant cells. A) The system typically consists of a Ti plasmid, into which the GOI is...(2020) Targeted delivery of nanomaterials with chemical cargoes in plants enabled by a biorecognition ...

...used to mutate the PAM sequence (an NGG sequence critical for Cas9 cleavage), thereby rendering the resulting...unique sites and allows for the introduction of specifically designed mutations (not limited to frame shift...temperature in the polymerase chain reaction." Analytical Biochemistry 209.2 (1993): 284-290. PubMed PMID...

...vitro, and what obstacles must be overcome for clinical success. ART and HIV-1 reservoirs HIV-1 infects...these methods represent exciting advances in preclinical HIV-1 research that should be pursued in animal... field, the lessons learned in pursuing one technological application can benefit many others and accelerate...

... incredibly important to the stability of the biological research enterprise going forward, in this post...Addgene), that need people who are data oriented, scientifically minded, and able to communicate complex information...underrepresented groups under their mentorship with the same critical eye they place on others within their labs - as...