...neurons in the adult dentate gyrus and olfactory bulb. Proc Natl Acad Sci U S A. 2013 Mar 19;110(12):E1152-61... 2008 Dec; 18(6): 617–623. Pubmed PMID 19349161.  2. Deshpande A, Bergami M, Ghanem A, Conzelmann KK, ...foundational later on when we studied biological processes, like how our stem cells are housed in specific...G-deleted RABV can divide and replicate (since these processes are mediated by other viral proteins), but the...

...digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org/10.1089/hgtb.2013.131 Lock,...cells, wait 2–5 days and then collect the supernatant and/or producer cells for further processing. What component...quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Human ..., R., & Kleinschmidt, J. (1999). Titration of AAV-2 particles via a novel capsid ELISA: Packaging of genomes... genomes can limit production of recombinant AAV-2. Gene Therapy, 6(7), Article 7. https://doi.org/10.1038...Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material. Human Gene Therapy, 21...over from the production process. For more details on the qPCR titering process, refer to the Addgene AAV...

...either (1) a lack of gRNA and/or Cas9 expression or (2) a lack of efficient target cleavage in cells expressing...is necessary to model your specific disease or process of interest. Do you want to: Generate complete ... method of delivery and the cell type. Before proceeding with your experiment, it may be necessary to ...

...severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus may acquire mutations to escape ... SARS-CoV-2 infects host cells through binding the human angiotensin converting enzyme 2 (ACE2) cell-surface...Pooled library to study antibody escape in SARS-CoV-2 Recombinant antibodies for aminoacyl tRNA synthetases...viral preps Mapping antibody escape in SARS-CoV-2 by mutational scanning By Seth Kasowitz As multiple...viral spike protein.   Figure 1. (A) SARS-CoV-2 RBD DMS Library generation and sequencing. From Starr... binding ACE2 is the target of most anti-SARS-CoV-2 antibodies. In the library, each plasmid encodes the...and a barcode, and is designed so that the SARS-CoV-2 spike RBD is displayed on the yeast cell’s surface...

...the image can be acquired more quickly. 2-Photon Microscopy 2-Photon Microscopy attempts to solve two ...Fluorescence microscopy. Nature methods 2, 910-919 (2005). PubMed PMID: 16299476. 2. Conchello JA, Lichtman JW. Optical...enhance spatial contrast (i.e. optical sectioning (2) or super-resolution (3). Fluorescence microscopy...photobleaching and a reduction in signal intensity. 2-Photon microscopes restrict excitation (and bleaching...can be seen exiting through the tissue. Therefore, 2-photon allows for deeper imaging into thick tissues...imaging objective that collects fluorescence. Like 2-photon microscopy, only a single focal plane of the...relying on raster scanning as is done in confocal or 2-photon microscopes. Total Internal Reflection Microscopy...

...comprehensive SARS-CoV-2 virus-human interaction network in which we expressed 26 of the 29 SARS-CoV-2 viral proteins...all became acutely aware of the threat of SARS-CoV-2. Like many in the science community, we’ve been motivated...identifying these potential human helpers of SARS-CoV-2 (especially the ones that are druggable) is a key ...just at the beginning of understanding how SARS-CoV-2 interacts with human proteins. For example, we used... joining in this worldwide fight against SARS-CoV-2. As we’ve all been affected in various ways by the...details. Our set of constructs comprises all SARS-CoV-2 viral proteins except for Nsp3 and Nsp16, and includes...is going on there!).  Find the Krogan Lab SARS-CoV-2 plasmids (Note: The Nsp6 construct is undergoing QC...

... be extracted from small bacterial cultures (i.e. 2-3 ml) with or without commercially available kits ...ratios measured at 260 and 280 nm should fall ~1.8 - 2 and give scientists an indication of purity versus...contaminants. The 260 and 230 nm ratios should fall ~2 - 2.2, and provide an indication of purity versus ...hydrochloride used during plasmid extraction. Figure 2: Use cloning grade DNA for direct cloning of your ...Transformation grade DNA  Transformation is the process of inserting exogenous DNA in a microorganism. ...contaminants that have been used during the purification procedure. Good indicators of DNA purity are the absorbance...transfection grade DNA. Transfection is the process of inserting an external plasmid in mammalian cells...

...determined by both codon bias and folding energy. Proc Natl Acad Sci USA 107:3645–3650 https://doi.org/...Hoogsten base pairs and wobble pairs.  Figure 2: (A) The codon wheel depicts the 64 codons that encodes...

...the addition of Cre recombinase (Figure 2). Figure 2: A knockout targeting vector designed to... an idea of the process, we’ll walk through an experiment designed to knock out exon 2 of a given gene...For recombination to occur in a cell, a minimum of 2 kb of sequence homology is required, but 6 to 14 kb...construct to your target cells. After recombination, exon 2 of the targeted gene will be removed from the chromosome...the GFP/NeoR cassette is inserted in place of exon 2. Thus, the targeted gene is disrupted (knocked out...recombination event will remove NeoR only and leave exon 2 floxed, as seen in line 4 of Figure 4. Since the loxP...Gene targeting takes advantage of this natural process to replace a targeted genetic locus with homologous...

...Biol. 2015. PubMed PMID: 25895059 Barnea, et al. Proc Natl Acad Sci U S A. 2008. PubMed PMID: 18165312...Science. 2013. PubMed PMID: 24136966 Nyerges, et al. Proc Natl Acad Sci USA. 2016. PubMed PMID: 26884157 ...generating protein chains or clusters. Veggiani, et al. Proc Natl Acad Sci U S A. 2016. PubMed PMID: 26787909...backbone, which expresses the gRNA from a Drosophila U6:2 promoter and Cas9 from the actin 5C promoter. Addgene...backbones for lentivirus LV 1-5 and retrovirus RV 2-5 were constructed. The authors then used GMAP assembly...engineering (MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate multiple single-strand...number of tandemly expressed gRNAs respectively) and 2) the size of the deletion. Smaller deletions generally...

... needing to work with a transfectable cell line. 2) They require larger expression vectors which are ...are also larger than their Cpf1 counterparts. Table 2. Cas9 Multiplexing Options  Multiplexing Method... Cpf1 expression vector.  See the graph in figure 2 for a comparison of editing frequency resulting from...Biotechnology 35.1 (2016): 31-34. Pubmed PMID: 27918548. 2. Zetsche, B., Gootenberg, J., Abudayyeh, O., Slaymaker...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell,163(3), 759-771. PubMed PMID...like Csy4, or the inclusion of processing signals, such at tRNAs, to process the array into functional crRNAs...Cpf1’s ability to process its own pre-crRNA arrays simplifies the crRNA cloning process. For cloning, Zetsche...

... H. J. (2009). Rats! Disease Models & Mechanisms, 2(5–6), 206–210. https://doi.org/10.1242/dmm.002733 ...that has defined our understanding of biological processes and developments. Model organisms often feature...history and characterization. Many proteins and processes naturally found in E. coli have been manipulated.... coli can be used as a model to relate these processes in more pathogenic bacteria (Blount, 2015). Rattus...McCusker and Gardiner, 2011). As many biological processes and signaling pathways that regulate regeneration...humans. Thus, understanding their life cycle and processes with their regenerative abilities is expected ...the most significant and fundamental biological processes. Many of their genetics and developmental patterns...

...illustrated in the schematic below.   Figure 2: gRNA target sequences (colored rectangles) are cloned... plasmids: This plasmid set allows you to express 2-4 gRNAs, with four being the ideal number. First you...the total number of gRNAs you wish to clone, from 2-7. For example, if you’re expressing 4 gRNAs, you’...gRNAs into spectinomycin-resistant plasmids pX330S-2 to pX330S-(last gRNA number). The 5’ most gRNA is ...Multiplexing Plasmids: These plasmids allow you to assemble 2-4 gRNAs through Golden Gate or Gibson Assembly. gRNAs...Multiplex Plasmids: These plasmids allow expression of 2-5 gRNAs in zebrafish. Custom destination vectors are...and a subsequent curing protocol that requires only 2-3 hours incubation. Kondo Lab multiplexed base editing...

...the resulting fraction is reported. Figure 2: Normalization with loading controls. Under each lane...control band is weaker in condition 1 than condition 2, the normalized values for the two conditions are ...opposite change in your loading control. Assumption #2: The loading control can be accurately quantified ...significantly lighter in “condition 1” than “condition 2.” By eye, the loading control appears about the same...) are smaller than the analogous green “condition 2” points by the exact same value. For the two solid...consider a different loading control. Assumption #2: The loading control can be accurately quantified ...factors we’ve covered in our previous blog posts (1, 2) on western blotting. If the abundance of your POI...

...transductions and use a range of reasonable MOIs. 2. Create a monoclonal Cas9-expression cell line. Especially...doing a screen. I made a few monoclonal lines (Figure 2) and I was surprised at how variable Cas9-expression...across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal cell lines generated... expression Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9, but I didn... appear green despite having active Cas9 (see tip 2). Pro-Tips 1. To quantify Cas9 activity using this...Western blotting against GFP expression, but I didn’t. 2. In the original publication they see GFP expression...and I got clear bands using the conditions above. 2. Design your primers so that the digestion site isn...

.... To understand why that is, let's look at Figure 2. The blue laser excites at a wavelength of 488 nm ... of PE's excitation spectrum (dotted line, Figure 2). The yellow/green laser, however, excites at 561 ... stronger — or brighter — signal.   Figure 2: Excitation and emission spectra for PE. The excitation...this example, I will describe a basic panel (Table 2, Figure 5) for a hypothetical experiment in the lab...efficiency (eGFP expression).     The first item in Table 2 is a standard and should always be included—the live...from the other fluorophores to avoid bleeding and 2) BV421 is a very bright fluorophore, which makes it...case the CD45 expression turns out to be weak. Table 2: A simple flow panel to detect live, transduced, and...

... that results in a polymerase error rate of up to 2% per nucleotide position. The “sloppy” PCR reaction...modifications: 1) increased concentration of Taq polymerase, 2) increased PCR extension time, 3) increased concentration...to synthesize a complementary mutant strand (step 2 and 3). Then the uracil-labeled template strand is...restrict site matter? Most of the time no, but there are 2 instances where it does. 1) When the backbone vector...ExoIII. See the below figure for an example like this. 2) When your gene of interest has a BbvCI site(s). If...saturation mutagenesis method are 1) ease of use, 2) time savings, and 3) ability to perform single site...PMID: 23766324. PubMed Central PMCID: PMC4409680. 2. Mccullum, E. O., Williams, B. A., Zhang, J., & Chaput...

...generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required for recombineering...generic lambda red recombineering experiment (Figure 2). In the following sections, key steps that differ...confirmation of recombinant clones.    Figure 2: Overview of using Lambda Red recombineering system...pairs of homology to the targeted insert site (Figure 2, top).  These primers are typically ~70 nucleotides...bacteria that has key MMR proteins knocked out or 2) specially design ssDNA oligos to avoid MMR: 1) E....will have more unintended changes to their genomes. 2) Designing ssDNA oligos that avoid MMR activation:...

...and Amy tell us a little about themselves. Chapter 2: What is Science in the News (SITN)? - SITN is a graduate...The SITN Editorial Process -Vini discusses the importance of the editorial process and peer-to-peer feedback...

...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems use...systems to carry out various functions. Class 2 Class 2 CRISPR systems rely on a single protein Cas effector...explore Class 2 CRISPR in more depth, the Jennifer Doudna lab maintains an encyclopedia of Class 2 CRISPR systems...systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https://doi.org...complex for antiviral defense) complexes, while Class 2 use single protein Cas effectors, like Cas9. The Cascade...1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible abundance, Class...components present in the Cascade complex. Figure 2: Schematic of a Cascade complex (blue subunits) with...