...manipulate, 2) they have long-term expression; and 3) they have limited toxicity. However, a key challenge...1x1011 vs 1x1012 viral genomes (vg) per mouse. Figure 3 shows a gross comparison of AAV.PHP.eB and AAV.PHP.S...transduction, measured via GFP expression, was determined 3 weeks later. Table was derived from figure 2e in Chan...transduction, measured via GFP expression, was determined 3 weeks later. Table was derived from figure 3b in Chan...PubMed PMID: 26829320. PubMed Central PMCID: 5088052. 3. Lentz TB, Gray SJ, Samulski RJ. Viral Vectors for...or the PNS, respectively.  Read on to learn more about these two new AAV capsids and how they can be useful...Important considerations when using AAVs Learn more about using AAV as a tool for gene expression in mammals...

...why is it necessary to have all 3 of the different binary systems above if they all behave very similarly...multiple genes at the same time.  The 3 binary systems listed above are orthogonal, meaning that they do... synthetic systems. Recent work has even used all 3 in a single modular vector for ease of use! Conclusion...313. doi: 10.1007/s00253-018-9485-4. Epub 2018 Nov 3. PMID: 30392122. Pfeiffer, Barret D., et al. “Refinement..., lineage tracing, and mosaic analysis.” Cell 141(3) (2010): 536-48. PubMed PMID: 20434990. PubMed Central...reagents for genetic manipulations.” Nat Methods 12(3) (2015): 219-222. PubMed PMID: 25581800. PubMed Central...can be used in the repressible manner described above, as well as the inducible manner detailed in our...

...length. It contains short promoter sequences at its 3’ and 5’ ends that are crucial cis-acting signals for...shortened version of the viral genome. It contains the 3’ and 5’ ends of the genome which are required for ...nonsegmented negative sense RNA viruses (reviewed in 2, 3). Other important components of the Zaire ebola virus...the viral polymerase initiates RNA synthesis at the 3’ end of the minigenome to produce the positive sense...Berlin Heidelberg, 2004. 1-41. PubMed PMID:15298166. 3. Hoenen, Thomas, et al. "Minigenomes, transcription...Microbiology, National Emerging Infectious Disease Laboratories.  She is interested in genome replication and...

...." F1000Research 3 (2014). Pubmed PMID: 25653845. Pubmed Central PMCID: PMC4304227. 3. Sauermann, Henry...positions within 5-7 years of earning their PhDs (3), showing that the ‘standard’ pathway into academia..., Eve. "Looking out for future scientists." eLife 3 (2014): e04901. Pubmed PMID: 25291257. Pubmed Central... the Jackson Laboratory for Genomic Medicine and a Senior Research Associate at the Labor and Worklife...increasing data collection about the scientific workforce, in particular about postdocs. Among other involvements...Research Symposium Boston 2015 – Academic Data and the Labor Market. For more information on the symposium as...generating too many trainees per PI” “Lack of awareness about how the system operates and functions” “Complete...

...plasmids ready for deep mutational scanning.     3 reasons to be interested in deep mutational scanning... Taq polymerase, 2) increased PCR extension time, 3) increased concentration of MgCl2, 4) increased concentration...synthesize a complementary mutant strand (step 2 and 3). Then the uracil-labeled template strand is degraded ...mutagenesis method are 1) ease of use, 2) time savings, and 3) ability to perform single site or multi-site saturation...Mutagenesis Protocols, pg. 103-109. PubMed PMID: 20676978. 3. Wrenbeck, Emily E., Justin R. Klesmith, James A. ... all desired codon changes (step 1 in the image above). A second universal primer is used to synthesize...first followed by the top strand. Both produced about the same number of positive clones. See Supplementary...

...the termini of the crRNA molecule (usually 1-3 of the first 3 bps are modified) for optimal stability and...crRNA also contains a ~20 bp repeat region on its 3’ end. This repeat region associates with the trans-activating...sugar ring, a popular stabilizing modification is 3’ phosphorothioate linkage, where a sulfur replaces...“I hope it isn’t degraded” when scientists talk about their precious RNA samples. RNA is inherently less...efficacy as well as the unnecessary stress of worrying about the status of your RNA. gRNAs modifications are ...Some of the RNA stabilizing modifications listed above were developed before CRISPR-Cas9, for use in applications...

...antigen. J Gen Virol., 73 (Pt 3), 653–660. https://doi.org/10.1099/0022-1317-73-3-653. PMID 1372038. Randall...comparable to GFP during early embryogenesis (Figure 3). mScarlet-I3 also offered clearer signals due to ...autofluorescent tissues like the intestine. Figure 3: Expression of fluorophore-tagged GOLG-4 through eight...disruption in Mycobacterium abscessus. J Bacteriol., 206(3), e0033523. https://doi.org/10.1128/jb.00335-23.  ...more feasible, the lab of Alessio Ciulli and collaborators developed CRBNmidi (Kroupova et al., 2024). ...stepwise, achieving mutation efficiencies of up to about 9% of colonies (102 to 104 times higher than traditional...Bentley Time to get flexible! Alex Dunn’s lab and collaborators have deposited a new mechanosensor called PILATeS...

... step involves digesting phage genomic DNA into 1-3 kb (approximately gene-sized) fragments so that these...bulk of resultant fragments falling between 1 and 3 kb results in optimal likelihood of capturing a whole...filter paper and saturate each with lysis solution (~3 ml each), pouring off any excess. When your protein...Expression Vectors in the FX Cloning System Kit  Step 3: Probe random phage DNA fragments with live host cells...PMID: 27536293. PubMed Central PMCID: PMC4971087. 3. Simpson, David J., Jessica C. Sacher, and Christine..." Chemical Reviews (2016). PubMed PMID: 27319741. 3. Simpson, David J., Jessica C. Sacher, and Christine...our planet, but we still know collectively little about how they infect and influence the evolution of their...

...crRNACpf1 Endonuclease size spCas9: ~4 kbsaCas9: ~3 kb ~3.9kb crRNA/gRNA length gRNA: ~100 nt crNA:... Lastly, an AAV vector was used to multiplex edit 3-4 genes in a primary culture of mouse neurons and ...and GFP.  Of these neurons, ~15% had indels at all 3 targeted loci.  See the chart below for a summary ...Endonuclease of a Class 2 CRISPR-Cas System. Cell,163(3), 759-771. PubMed PMID: 26422227. PubMed Central PMCID...targets compared to Cas9.  Read on to learn more about Cpf1 multiplexing.  For an in-depth review of Cpf1... Addgene. If you have any questions or thoughts about Cpf1 multiplexing, leave them in the comments below...

...Recombinant DNA by CcdB.” Biotechniques. 1996 Aug;21(2):320-3. PubMed PMID: 8862819. 2. Bahassi, EM., et al. “F ...Microbiol. 1995 Mar;15(6):1031-7. PubMed PMID: 7623659. 3. Bernard, P., et al. “Positive-selection vectors using...DNA cleavage by gyrase.” J Mol Biol. 1993 Dec 5;234(3):534-41. PubMed PMID: 8254658. 5. Bernard, P. and ...DNA-topoisomerase II complexes.” J Mol Biol. 1992 Aug 5;226(3):735-45. PubMed PMID: 1324324. 6. Tam, JE. and Kline...in modern cloning, and why should you learn more about it? Read on to find out how ccdB can make your cloning...of this system for enhancing cloning efficiency about 20 years ago and developed cloning vectors to harness...

...Scientific reports 3 (2013). PubMed PMID: 24284873. PubMed Central PMCID: PMC3842082. 3. Mashiko, Daisuke...pCAG-EGxxFP target plasmid contains overlapping 5′ and 3′ EGFP fragments under the ubiquitous CAG promoter....classified into four groups (4; brighter than control, 3; same as control (Cetn1/sgRNA1), 2; darker than control...sequence the resulting plasmid. It can take roughly 3 months from the day you decide to do a gene modification...the pX330 plasmids used in the validation assay (above) and it is just as effective (Mashiko et al., 2014...Engineering with CRISPR Validate Your Genome Edit Learn about Non Homologous End Joining and Homology Directed...

...Topic Page Learn about Non-Homologous End Joining Learn about Base Editing Read 3 Tips to Improve ... break is resected by nucleases to create a ssDNA 3’ overhang. This will serve as both a substrate for... repair (DSBR) In the classical DSBR pathway, the 3’ ends invade an intact homologous template to serve...the non-invading strand at the other DSB end. The 3’ end of the non-invasive strand is elongated and ligated... donor DNA. Richardson CD, et al. Nat Biotech. 34(3); (2016). PubMed. Efficient homology-directed repair...

...the CEO of 3-D Molecular Designs.     Additional Resources on the Addgene Blog Learn All about CRISPR Use...MSOE center for BioMolecular Modeling and the CEO of 3-D Molecular Designs. Have you ever held your favorite...models were useful in that they not only traced the 3-D path of the protein’s backbone in three-dimensional...arginine 53 really is right there, at the end of helix 3…just like we said it was”.   Seeing is believing. ...Until then, you can only wonder what I am talking about. How do researchers use physical models of protein...students work closely with local researchers to learn about their research while creating physical models of...model is an effective teaching tool. You can read about Jacob’s initial reaction to this model on his blog...

...field, (2) that you have three 3 years of research experience in the field, (3) that you have an offer of ...applications and approved 91,023 during the first 3 quarters of 2017. As an international researcher and...well positioned to advance the proposed endeavor; (3) The foreign national’s proposed endeavor will be ...for this category. To qualify, you need to satisfy 3 out of the 10 criteria listed below. An international...of the institute. For the EB-1B, you need to prove 3 threshold requirements: (1) that you have international... you must show that your company employs at least 3 full-time people engaged in researcher activities.... data and the list goes on and on. As you think about your career (and your future in general), keep in...

...three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required for recombineering with...DNA introduced into the E. coli. Exo: Exo is a 5’→3’ dsDNA-dependent exonuclease.  Exo will degrade linear... 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the dsDNA was short enough, a ...the translated protein.  These changes can be 5’ or 3’ of the desired change.   Table 1: Overview of ds-... integrated defective prophage 2) from a plasmid, 3) from mini-λ or 4) from the lambda red phage itself...PubMed PMID: 10811905.PubMed Central PMCID: PMC18544. 3. Murphy, K. 2016. λ Recombination and Recombineering...currently a research scientist in the Department of Laboratory Medicine at the University of Washington. She...

...15;8(3):535-42. PubMed PMID: 23256573. Additional Resources on the Addgene Blog Learn More About the ...yeast as an alternative source of essential omega-3 fatty acids complementing vegetable oil, a somewhat...PMID: 27984002. 2. Bilan, Dmitry S. et al. “HyPer-3: A Genetically Encoded H2O2 Probe with Improved Performance...The Addgene-Seeding Labs plasmid grant is a collaborative endeavor providing plasmids to researchers in... of Zimbabwe, Zimbabwe Dr. Idah Sithole-Niang (above, left) is an Full Professor in Biochemistry with... CRISPR effect observed in bacteria. Learn more about the different CRISPR applications in plants.     ...Rodrigo Arturo Rivera Solís, Associate Professor (above, far right), thrives to make affordable biosensors...

...mutations in RPE65 (n=3), showed significant improvements in vision over a period of 1 to 3 years – the treatment...neutralizing antibodies against some AAV types, AAV1, 2, 3 and 5 are thought to be as high as 70% among the human...PMID: 20463753. PubMed Central PMCID: PMC3152950. 3. Jacobson, Samuel G., et al. "Improvement and decline...Vectors Featured Topic Page Learn about CRISPR-AAV vectors Learn about Addgene AAV Quality Control Process...viral infections. She is particularly passionate about HIV cure research and science communication.     ...

...Figure 1, large sequences corresponding to exons 1 and 3 of the targeted gene have been cloned into the vector...Neomycin resistance (NeoR) gene between exon 1 and 3 of the targeted gene. Upon recombination, the GFP/...-PGK-Puro from the Jaenisch lab.   Figure 3: A knockout/knock-in targeting vector designed to ...resistance marker using Cre recombinase. Since there are 3 loxP sites, recombination can occur in multiple ways...inserted GFP is expressed (knocked in). As seen in the above example, you can remove a floxed resistance gene...Get the Basics of Homology Directed Repair Read about Generating Knockouts with CRISPR through Non-Homologous...Expression Vectors Find Cre-Lox Plasmids Learn More about CRISPR  ...

...sequence (Zong et al., 2018) (see CBE-3 in Figure 3). Figure 3: Activity windows of plant base editors...position 4-8 counting the ‘NGG’ PAM as 21-23 (Figure 3). If the target ‘A’ is closer to the PAM, i.e., positioned... 353.6305 (2016): aaf8729. PubMed PMID: 27492474. 3. Gaudelli, Nicole M., et al. "Programmable base editing...gene, Xw, except for a single base. If you heard about CRISPR, one question probably comes to mind: can...compatibility All of the base editors mentioned above are SpCas9 nickase-based and depend on the presence... HDR methods for rice crop improvement in the laboratory of Yinong Yang at PSU.   References 1. Komor,...The CRISPR-Cas Revolution, Cold Spring Harbor Laboratory (2018). Additional resources on the Addgene blog...

...more than 3-4 colors due to the tandem integration of multiple copies of the construct (about 8 in Brainbow...colorful technology. Optimizing the system: Brainbow-3 Although Brainbow provided neuroscientists with the...Looger LL, Simpson JH. Nature Methods. 2011 Mar;8(3):253-9. doi: 10.1038/nmeth.1566. Epub 2011 Feb 6. ...Dickson BJ, Salecker I. Nature Methods. 2011 Mar;8(3):260-6. doi: 10.1038/nmeth.1567. Epub 2011 Feb 6. ...Beaurepaire E, Morin X, Livet J. Neuron. 2014 Feb 5;81(3):505-20. PubMed.   ...with the goal of overcoming the limitations listed above. First, they screened a variety of fluorescent proteins...method has become very useful in lineage tracing; “Zebrabow” was used to trace the development of the corneal...