...Part 2: Building Community  My editor’s choice pick for this month is “Grad School Advice Part 2: Building...Options for CRISPR Genotyping Grad School Advice Part 2: Building Community Engineering the Plant Genome Using...graduate school classmates, Ben Vincent and Niroshi Senaratne. When we recorded, Ben and Niroshi, were finishing...

...for the 2+ million DHS sites. See below for further details. DNA is a 3D Fractal Figure 2: DNA compaction...: 1. Find Enhancers that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the ...NANOG enhancers 3. CRISPR out the enhancers from step 2. See which ESCs die or differentiate Step 3 reveals...expression for NANOG enhancers, we’re ready for step 2: design a CRISPR screen to disrupt these enhancers...to CRISPR out the enhancers we identified in step 2. The result is a heterogenous population of stem cells...determination was established with Bowtie2 using the parameters first described in Kearns et al.: bowtie2 -f ...

...NGG)  Editing window ~2 nt  85175  pBK-EE-BE3  SpCas9 (NGG)  Editing window ~2 nt  85176  pBK-YE2-...quantum yield of 0.54) and mScarlet-H with M164H (2-fold improvement in photostability compared to mScarlet...SpCas9 base editors with editing windows as small as 1-2 nucleotides! To reduce off-target effects associated...pBK-YE2-BE3  SpCas9 (NGG)  Editing window ~2 nt  85177  pBK-YEE-BE3  SpCas9 (NGG)  Lower on-target activity...activity than BE3; editing window ~1-2 nt  87438  pET42b-HF-BE3  HF-Cas9 (NGG)  Very low off-target activity...Ladders Are Awesome Tools: They are comprised of 2 plasmids, pPSU1 and pPSU2, that can be amplified affordably...libraries The Gross Lab at UCSF recently deposited 2 Bacillus subtilis libraries with Addgene – the B. ...

...digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org/10.1089/hgtb.2013.131 Wang,...(see maps section for images).     Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre...Digital PCR Method. Human Gene Therapy Methods, 29(2), 96–103. https://doi.org/10.1089/hgtb.2017.198   ... lentiviral preparations (“-LVC” catalog items) are generated from lentiviral preparations (described ... to the cleared viral preparation pool. In addition, the final viral preparation undergoes PCR with primers...specific virus in order to generate high quality preparations. After production, all virus preps are titered...plasmid encoding adenoviral helper sequences. AAV preparations are purified by iodixanol gradient ultracentrifugation...

...University of Manitoba due to their Biosafety Level-2 or 2+. Please Note: Despite the fact that AAVs themselves...long and stable expression (up to 9 months in mice [2]). Low Immune Response: Compared to other viral vectors... location. Location (1): What tissue? Location (2): Where in the tissue/which cell type? Location (3...may be sufficient to overcome the transport issues. 2. Failure to plan (for storage) Is planning to fail...Find Ready-to-Use Retrograde AAV Preps at Addgene 2) Comparison of genetically engineered receptors (i.e...Therapy 15.10 (2007): 1740-1743. PubMed PMID: 17882145. 2. Zincarelli, Carmela, et al. "Analysis of AAV serotypes... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain." PloS one 8.9 (...

...(bell curve) or skewed (see Fig. 2). Assumptions – characteristics of a data set; the ‘rules’ a statistical...    Fig. 2: Examples of data distribution profiles.   Parametric tests  Statistical tests... positive is much higher than if you only look at 2 or 3 pairwise comparisons. Similar to family-wise ...statistical facets of reproducibility. Adv Physiol Educ. 40(2): 248-252 (2016). 0.1152/advan.00042.2016 Finch, W...Simulation Study. Front. Appl. Math. Stat. Volume 2 (2016). doi.org/10.3389/fams.2016.00002  ...categories: parametric and non-parametric. In this blog we will be discussing parametric tests. Parametric tests...requirements that need to be met for a parametric test are as follows:   Parametric test assumptions: Distribution...

...Tris-tricine 30 V, 1 hour or 100 V, 1–2 hours Good separation, quality, and stability Limited protein..., which will allow clear separation of proteins of different sizes.  Table 2: Recommended gel percentages.... The most common options are 4 °C overnight or 1–2 hours at room temperature (RT). Most people choose...overnight at 4 °C and their secondary antibody for 1–2 hrs at room temperature, which allows them to reduce...Tris-glycine 6–400 kDa Tris-glycine 100 V, 1–2 hours Easy and cheap to handcast Short shelf-life...are consistent and stable at room temperature for 1–2 years and are available in formulas specific to your...response over a broad dynamic range that typically spans 2–5 orders of magnitude, which includes a wide dynamic...

...improving editing accuracy by preventing re-editing and (2) optimizing “cut-to-mutation distance”. Furthermore...also takes into account that HDR is rare, usually 2-5% of the edited cells in our hands, these combined...screen a few hundred clones to find the right one Step 2 – Edit the cells a second time using a repair template...Additional CRISPR Resources on Addgene's CRISPR Guide Page 2. Optimizing "cut-to-mutation distance"  Even though... wisely (and let them cut close).   Figure 2: Relationship between cut site to mutation distance...PMID: 23287718. PubMed Central PMCID: PMC3795411. 2. Mali, Prashant, et al. "RNA-guided human genome engineering...incorporated during HDR. We decided to systematically characterize this phenomenon and found a general relationship...

...guide expression and dsDNA breaks. Figure 2: Non-targeting vs. safe-targeting guides. The genome-wide...target all protein-coding genes present in the genome; 2) non-targeting guides (red), which target no sequence...guides that had zero mismatch off-targets. Guides with 2-bp mismatch off-targets were only toxic if they had...tolerated than mismatches more distal to the PAM, and 2) guides with high GC content have greater off-target...PMID: 28474669. Pubmed Central PMCID: PMC5424143. 2. Tsai, Shengdar Q., et al. "GUIDE-seq enables genome-wide...effects. The flaws of RNAi screens are well characterized and strategies exist to control for these faults...also reproduced several previously observed characteristics that influence gRNA off-target activity: 1)...

... this problem? (2-3 sentences) What will be the focus of your first grant? (1-2 sentences) ...of scientific success is publications and grants. 2. Write succinct and easy to understand documents A...consideration. 3. Present an easy to follow seminar See item 2. Attendees will include grad students (that are solicited...people would agree is important to understand. (1-2 sentences). For example: Distinguishing correctly ...emphatically deny that I have a magic formula to guarantee a reader the faculty job of his/her dreams. There...pitch that requires reading two pages or even a paragraph to get to the punch line is a terrible sales pitch...

...derived from direct comparative analysis of microbial opsins. Nature methods. 2012;9(2):159-72. doi: 10.1038...commonly used in optogenetics: 1) electrophysiology and 2) behavioral experiments. I’m not an electrophysiologist...choice in the brain region you wish to stimulate 2) Fabrication and implantation of the optical fiber...thoroughly elsewhere (here and here for instance) [2], [3]. Karl Deisseroth of Stanford University , one...reviews for a discussion of the different variants [1, 2, 4]. And, of course, the best way to learn about the...monitor expression at various time points (e.g. every 1-2 weeks over 8 weeks) in order to determine the optimal...(4):251-66. doi: 10.1038/nrn3171. PMID: 22430017. 2. Fenno L, et al. The development and application of...

... Multiple Introns.” Plant Communications, vol. 2, no. 2, Elsevier BV, Mar. 2021, p. 100135. Crossref, ...Constructs.” PLoS ONE, edited by Jean Peccoud, vol. 6, no. 2, Public Library of Science (PLoS), 18 Feb. 2011, p... vol. 5, no. 10, American Chemical Society (ACS), 2 May 2016, pp. 1059–69. Crossref, https://doi.org/10.1021...of Plant Genomes.” The Plant Genome, vol. 16, no. 2, Wiley, 9 Mar. 2023. Crossref, https://doi.org/10.1002...Crossref, https://doi.org/10.1186/s12870-020-02388-2. Stuttmann, Johannes, et al. “Highly Efficient Multiplex...Crossref, https://doi.org/10.1038/s41598-019-46171-2. Resources on Addgene.org Modular Cloning (MoClo....6b00337. Lee, Michael E., et al. “A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly...

...these topics. Listen to Part 1 Here Listen to Part 2 here! Time : Topic 0:00 - 0:50 : Intro 0:51 - 3:08...series, we continue our conversation with Niroshi Senaratne and Ben Vincent from the Biological and Biomedical...Biology, and he tweets @benjvincent.     Niroshi Senaratne is a graduate of the PhD program in Genetics and...

...detect your targeting vector or gene of interest. 2. Breed your parental mice to generate F1 Once you’...Instead, genetic engineering should be used to separately introduce each mutation independently into different...parental generation, they are likely to stay on separate chromosomes in future generations as well, a concept...

...sequence YFP Thorben Dammeyer 54520 mCherry-Peroxisomes-2 Peroxisome Peroximal Targeting Signal 1 (PTS1) mCherry...Centrin2 Centrioles (dependent on cell cycle) Centrin-2 EGFP Erich Nigg 41151 pEGFP Cep170 C-term Centrioles...GolgiCFP Golgi apparatus eNOS(1-33) CFP Alexandra Newton 36205 pmTurquoise2-Golgi Golgi apparatus B4GALT1(1...10 Golgi apparatus MannII mGold Francois St-Pierre 85050 pmScarlet-i_Giantin_C1 Golgi apparatus Giantin...pmScarlet-H_Giantin_C1 Golgi apparatus Giantin mScarlet-H Dorus Gadella 85048 pmScarlet_Giantin_C1 Golgi apparatus Giantin ...Giantin Golgi apparatus Giantin mTurquoise2 Dorus Gadella 98880 mNeonGreen-Giantin Golgi apparatus Giantin ...targeted to the same structures as these well-characterized proteins. General Structures The cell contains...

... analogs such as 2-fluoroadenine (2-FA), organisms with functional APT will convert 2-FA to cytotoxic ...selection of plants undergoing RNAi, and LIIbeta F 1-2 RNAi, which allows assembly of intron-spliced hpRNAs...nucleotides, resulting in death. In the presence of 2-FA, potent gene silencing of APT is required for survival...target of RNAi is expressed as inverted repeats separated by a loop region that facilitates the formation...the RNAi trigger delivering the RNAi trigger characterizing the RNAi phenotype. Although all these steps...amiRNAs based upon miRNA precursors of rice and Arabidopsis species, respectively. Short hairpin RNA (shRNA...plants, such as in the model flowering plant Arabidopsis thaliana (Li et al., 2013; Zhang et al., 2018...

...then packaged into AAV’s protein shell, or capsid; 2) the pHelper plasmid which expresses adenovirus genes...takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR run and data analysis. Refer to...quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Human ...therapy methods, 23 1, 18-28. PubMed PMID: 22428977. 2. Choi, V.W., Asokan, A., Haberman, R.A., & Samulski...concentrations of iodixanol to separate out contaminants from an impure AAV preparation. 15%, 25%, 40%, and 60%... doesn’t get too hot because then the PEG will separate into two phases. If this happens, the solution...

...Carballosa, et al, (2015) eNeuro, March/April 2015, 2(2) e0001-15.2015. Pubmed. Resources: http://openspim.org...orthogonal-plane fluorescence optical sectioning (OPFOS) (2). Arne Voie, David Burns and Francis Spelman focused...in LSFM. In LSFM the laser light sheet, typically 2-6  microns, illuminates only one thin plane of the...successful attempt to render fixed anatomical preparations transparent was done by Walter Spalteholtz (...Durchsichtigmachen von menschlichen und tierischen Praparaten. Leipzig: S. Hierzel.
 J.A Dent, A.G. Polson,...Pubmed. K. Chung, J. Wallace, S.Y. Kim, S. Kalyanasundaram, A.S. Andalman, T.J. Davidson et al. (2013)...

...Cas9 from Streptococcus pyogenes.   Figure 2:  Crystal structure of S. pyogenes Cas9 in the apo...NUC and REC lobes laying open like a book (Figure 2). The portion of the CTD that recognizes the PAM is...it doesn’t appear in the crystal structure (Figure 2) at all. When this crucial recognition region is disordered...regions, so crystal structures like those in Figures 2 and 3 don’t show these regions, even if they are present...itself at the bottom of the image. Stem loops 1 and 2 extend horizontally across both lobes. The guide RNA...is required for Cas9 nuclease function. Stem loops 2 and 3 are not strictly necessary for in vitro cleavage...open conformation, but highly flexible, as in Figure 2. The guide RNA is bound primarily by the REC lobe,...

...listed in Table 2. For example,  chaining-based cloning methods require that the separate modules combined... TagBFP 399nm/ 456nm histone 2B (H2B) Chromatin 2 Cerulean 433nm/ 475nm Lyn-tag (derived from the tyrosine-protein...based (chaining-based) cloning. The modules (Figure 2) are contained in individual plasmids and are each...suitable for combining endogenous sequences. Table 2: High Throughput Cloning Strategies Method Technology...PMID: 20205762. PubMed Central PMCID: PMC2822740. 2. Engler C, Kandzia R, Marillonnet S. A one pot, one...