...editing from ~30% in control samples to 0-10% in Acr samples. Acr proteins can also be used to prevent dCas9...might not be as effective. This is where Acr proteins come in. Acr proteins are small enough that they could...specific site. 2. Temporal, spatial, or conditional control of CRISPR activity Regulating Acr proteins by..., 2018). For example, AcrIIA4 mentioned above indicates that it’s the fourth Acr protein discovered that...in 2012 (Bondy-Denomy et al., 2012). Anti-CRISPR (Acr) proteins are phage-derived small protein inhibitors... the CRISPR-Cas immune system of bacteria. These Acr proteins were originally found in type I CRISPR-Cas...Cas9, Cas12, and Cas13 (Marino et al., 2020). Some Acr proteins are specific to a particular Cas protein...

... achieved this by fusing the anti-CRISPR Acr protein, AcrIIA4, to a photo-sensitive LOV2 domain from A...the light-inducible heterodimerizing cryptochrome 2 (CRY2) and calcium and integrin-binding protein 1 ...inactive, dead Cas9 protein (dCas9) fused to CIB1; and 2) the activator - the CRY2 photolyase homology region... fused to both the N- and C-termini of dCas9; and 2) CRY2FL-VP64, a fusion of full-length CRY2 and the...researchers saw an 11-fold increase in mRNA levels after 2 hr and a 400-fold increase after 30 hr. The system...authors fragmented Cas9 into N-terminal (residues 2-713, N713) and C-terminal (residues 714-1368, C714...nMagC714 instead of nMagHigh1-C714, generating paCas9-2. This change did not significantly alter the system...

...same site, is 2’-fluoro (2’-F), which substitutes a fluorine for the hydroxyl group at the 2’ position. ...most common of these is 2’-O-methylation, where a methyl group is added to the 2’ hydroxyl group of the...them together, for example phosphorothioates with 2’-O-methyl modifications, proved more stability than...generated by introduction of a single photocleavable 2-nitrobenzyl linker in the 20-bp targeting region of... the crRNA or the tracrRNA. Of course, in a single guide system (crRNA and tracrRNA as a single molecule...samples. RNA is inherently less stable than most macromolecules used in scientific research such as DNA or ...region associates with the trans-activating crRNA (tracrRNA) which interacts with the Cas9 protein. These ...

...antibody has an antigen binding site for a total of 2 binding sites per antibody. C) IgG’s are big enough...enough that multiple labels (green circles in figure 2 below) can be attached to each antibody. But being...displacement Require separate incubation of 1° and 2° Must use different species and/or different IgG ...Greater resolution due to lower label displacement 2° incubation can be skipped by pre-binding to 1° ...a fluorescent tag and the target antigen to about 2 nm, which can lead to higher resolution for super-...subclass.   Express the Nanobody In Bacteria: Table 2 also has a link to the plasmid you’ll need to express... Find plasmids from this publication at Addgene. 2. Bates, M., Böhning, M.I., Chug, H., Görlich, D., ...

...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Figure from Zetsche et al., 2015...2015. How was Cpf1 found and tested? Class 2 CRISPR systems, including the type II Cas9-based system...employed by class 1 systems. A putative new class 2 nuclease, Cpf1 (CRISPR from Prevotella and Francisella... the 3’ end of the target sequence. Figure 2: Effects of (A) double and (B) single base pair gRNA-target...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227... Find plasmids from this publication at Addgene. 2. Zetsche, Bernd, et al. "Multiplex Gene Editing by...and requires only one RNA rather than the two (tracrRNA and crRNA) needed by Cas9 for cleavage. In certain...

...monoclonal lines (Figure 2) and I was surprised at how variable Cas9-expression was across the different cells...transductions and use a range of reasonable MOIs. 2. Create a monoclonal Cas9-expression cell line. Especially...cells (Figure 3).   Figure 2: Cas9 expression in monoclonal cell lines generated from A549 cells ... 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...

...IVT sgRNA in the lab. Figure 2: Cas9 RNP complexed with 2-part cr:tracrRNA tags genes more effectively ...approach (Dewari et al. 2018 and Figure 2). Not only is the cr:tracrRNA approach highly efficient, it is also...into stem cells. Cells are allowed to recover for 2-5 days and then analyzed for tagging efficiency using...cell suspension, transfect and let cells recover for 2-5 days.Note: We use the Nucleofection method for delivery...success was ~%50. If you get low knock-in efficiency (<2% positive by ICC for the tag) or do not see any cells...PMID: 28096221. PubMed Central PMCID: PMC5312033. 2. Dewari PS, Southgate B, Mccarten K, Monogarov G, ...days. The crRNA and tracrRNA are annealed on a PCR block and the annealed cr:tracrRNA is then mixed with...

...   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...

... 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...CRISPR Nucleases   Cas9 Cpf1 Components crRNAtracrRNACas9 crRNACpf1 Endonuclease size spCas9:...gRNA length gRNA: ~100 nt crNA: ~42 nt Requires tracrRNA? Yes No dsDNA cleavage Blunt end 5' overhang...

...Table 2 for more details on the effect of pH on the fluorescent properties of Rosella.   Table 2: Effect...in yeast. Autophagy,4(2), 205-213. doi:10.4161/auto.5331. PubMed PMID: 18094608 2. Sargsyan, A., Cai, J...environment like the vacuole or lysosome. See Table 2 for a summary of what color fluorescence Rosella emits...fluorescence accumulates in the vacuole. See Figure 2 for an example of what fluorescence looks like for...unless autophagy is induced. Then non-selective macroautophagy of the cytosol leads to accumulation of Pho8Δ60.... E. (2015). Rapid parallel measurements of macroautophagy and mitophagy in mammalian cells using a single...

...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. ...

...for retrograde transport has been found for AAV1, -2, -5, -7, -8 (Taymans et al., 2007), AAV1 (Hollis et... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One 8:e76310...reveal novel hippocampal CA2 circuits. Nat Neurosci 17(2), 269–279. PubMed PMID: 24336151. PubMed Central PMCID... Mechanisms of Motility and Regulation. Neuron 84(2), 292–309. PubMed PMID: 25374356. PubMed Central PMCID...transduction efficiency of recombinant AAV serotypes 1, 2, 5 and 8 in the rat nigrostriatal system. J Neurochem...analysis of adeno-associated viral vector serotypes 1, 2, 5, 7 and 8 in mouse brain. Hum Gene Ther 18, 195–...retrograde access to projection neurons. Neuron 92(2), 372–382. PubMed PMID: 27720486. PubMed Central PMCID...

... 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...

...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...monitoring of neuronal activity across substantial 3D volumes. In addition to 2-photon microscopy, lightsheet...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...

...Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1, but they require both a tracrRNA and ...however cutting efficiency of Cas13a is reduced when 2 mismatches are present. The protospacer flanking sequence...PMC5127784. Find plasmids from this paper at Addgene. 2. East-Seletsky, Alexandra, et al. "Two distinct RNase... and functional characterization of diverse class 2 CRISPR-Cas systems." Molecular cell 60.3 (2015): 385...PAM (PFS) Cleavage Mechanism Cas9 HNH, RuvC TracrRNA, crRNA DNA 5' NGG Specific blunt ended DSB...endogenous CRISPR/Cas9 system, Cas9 uses both a tracrRNA and crRNA to facilitate binding and cleavage of...

...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...

...month, from 1/22/20–2/23/20, where there were zero plasmid requests. Afterwards, from 2/24/20–3/15/20 (the...Each paper has between 1–32 plasmids and a total of 2–2,590 requests. We received plasmid requests from ...observed between Jan. 1 and Mar. 1, 2020. Figure 2: A simple approximation of the effect of COVID-19 ...However, it is evident that if we believe that 1/22/20–2/23/20 are the more accurate dates of lockdown in China...in the rate of scientists placing orders per day across regions, with Europe most impacted and China least...in the rate of scientists placing orders per day across regions. Our investigation of model fit revealed...

...of conventional gene targeting is lowered to about 2-3 months. But certain models remain more difficult...included in the final ssDNA template. Figure 2: Overview of ivTRT to generate an ssDNA repair template... floxed conditional allele requires you to target 2 loxP sites to intronic regions 0.5 - 0.8 kb apart....Typically, Easi-CRISPR works best with insertions of 2 kb or less. Luckily, most of the commonly used cassettes... induction systems (ttA or rtTA) are between 1 to 2 kb in length. There are plenty of Addgene plasmid ...create the ssDNA construct as described in Figure 2.  You can also use Addgene plasmids to purify Cas9... the separated guide RNA components (crRNA and tracrRNA) for maximum Easi-CRISPR efficiency. Easi-CRISPR...

...possible when each of the XFP genes is introduced at ~2 copies per cell per color, following a Poisson distribution...(prepared with Addgene #104110 - #104112) (Figure 2). We’ve also created constructs with containing chemical...AAV titer - The XFP genes have to be introduced at ~2 copies per cell per color to achieve the highest color...electroporation. Then at your preferred time points, sacrifice the animals and fix the samples. When you use ...AAV titer for the best results. The timing of sacrifice when using Tetbow AAVs - You will have to wait...

...or non-homologous end joining (see Figure 2).  Figure 2: Schematic representation of integration...due to insertional mutagenesis (Howe et al., 2008). 2. Transient expression of transgenes/shRNAs If you ...vectors: progress and applications. Gene Therapy, 17(2), 150–157. https://doi.org/10.1038/gt.2009.135. PubMed...efficiently during infection. Journal of Virology, 70(2), 721–728. https://doi.org/10.1128/JVI.70.2.721-728.1996...molecular biology for over three decades. Widely used across various research applications, they have become...lentiviral vectors, the lentiviral genome is distributed across multiple plasmids. For simplicity, we’ll use the...