...of other Cas proteins found in the so-called class 2 CRISPR-Cas systems. There are 24 variants of the cas14...the presence of infectious organisms (ex: SARS-CoV-2) and genetic mutations. By providing a gRNA specific...Last updated Apr 17, 2020. Before being adapted by scientists to edit the genome of virtually any organisms...on this planet, CRISPR-Cas systems were merely adaptive immune systems that provide bacteria protection...

.... 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...

...Homo line 2) or wild type (WT), two weeks after herbicide was applied to both plants. Adapted from Vu et... CLIPf tag and a C-terminal SNAPf tag; see Figure 2), the authors developed a cost-effective expression... when associated with microtubules. Figure 2: CLIP-SNAP fusion proteins for single-molecule FRET... B) Protein expression and labeling procedure. Adapted from Smith et al. 2024 under a CC-BY 4.0 license...(C) is indicated; red line is exponential fit. Adapted with permission from Hao and Lee et al., 2024. ...non-neurodegenerative diseases (Diabetes & cancer). Image adapted from Wu and Wang 2024 under a CC-BY 4.0 license...

...for controlling neuronal activity, channelrhodopsin-2 (ChR2), a blue light-activated cation channel, from...used in tandem with the opsin.      Figure 2: Diagram of excitatory (ChR2) activation by light ...) domains, phytochrome B (PhyB), and cryptochrome 2 (CRY2) (Zhang & Cui, 2015). When stimulated with light...comparative analysis of microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 References...comparative analysis of microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 Nagel...Hegemann, P., & Bamberg, E. (2003). Channelrhodopsin-2, a directly light-gated cation-selective membrane ...intracellular signaling pathways. Trends in Biotechnology, 33(2), 92–100. https://doi.org/10.1016/j.tibtech.2014.11.007...

... 25708933. 2. Cooper, Callum J., Mohammadali Khan Mirzaei, and Anders S. Nilsson. "Adapting drug approval... is also gaining a lot of new traction lately (1, 2), depends on knowledge of the structure(s) a given...per plate) across 20 fresh selective plates. Step 2: Express random phage DNA fragments in E. coli Now... biology 34 (2015): 69-77. PubMed PMID: 26275959. 2. Weigele, Peter, and Elisabeth A. Raleigh. "Biosynthesis...moved from the University of Alberta in Edmonton, Canada, to the University of Georgia, USA) has developed...

... Figure 2: Strategy for assembling barcoded combinatorial gRNA libraries. (Adapted from Wong et ...CombiGEM lentiviral vector backbone pAWp12 (Figure 2). Cloning from the pAWp28 storage vector to the pAWp12...PMID: 25114216. PubMed Central PMCID: PMC4151723. 2. Doench, John G., et al. "Optimized sgRNA design to...for assembling the barcoded gRNA library pool. (Adapted from Wong et al., 2016). With a list of gRNA...

... than control, 3; same as control (Cetn1/sgRNA1), 2; darker than control, 1; very dark). Brighter fluorescence...you can hold off on genotyping until the pups are 2-4 weeks old and use the ear tag method (creating different...PMID: 24253446. PubMed Central PMCID: PMC3875854. 2. Mashiko, Daisuke, et al. "Generation of mutant mice.../Cas9 for gene editing has expanded since its adaptation for use in mammalian cells in 2012-2013. Researchers... gene (although it has been quite successfully adapted for use in screening experiments as well). Therefore...fluorescence indicates a more successful gRNA design. Adapted from Mashiko et al., 2014. Not all gRNA’s are...chance of contamination with RNase leading to degradation that can set you back days. Working with plasmid...

...and a source of sdAbs (Figure 2) (Xu, et al., 2021).      Figure 2: Genetically modified mice offer... from camelid mice and llamas neutralize SARS-CoV-2 variants. Nature 595:278–282. https://doi.org/10.1038...single-domain antibodies. Image created in BioRender and adapted from Xu, et al., 2021.   The single-domain ...

... systems to engineer better and faster ABEs (Fig. 2). These systems allow for rapid continuous protein...and less stringent phage selection. Figure 2: Phage-assisted evolution of base-editing activity...testing numerous TadA variants, they identified TadA-8e, which edits 590-fold faster than TadA-7.10. It is ...deoxyadenosine deaminase, TadA-7.10 (Gaudelli et al., 2017). The idea was to evolve a TadA-7.10 version with ...evolution of TadA variants with much higher base editing activity compared to the original TadA-7.10 enzyme...additional mutation (V106W) into TadA-8e and the resulting ABE8e(TadA-8e V106W) was as efficient in on-target...codons within T7 RNAP. A phage encoding the deaminase TadA-7.10 A mutagenesis plasmid controlling the phage...

...method is the Standard Curve method. See Table 2, (adapted from Wong, 2018) for a comparison of different...phase   Table 2: A comparison of different qPCR analysis methods. Table adapted from Wong, 2018.  ...indicating primer dimers. (c) A double peak indicating 2 amplicons. Image credits (respectively): Zuzanna K...details for reproducibility’s sake!   Fig. 2: Setting up triplicates in multiple configurations... for RNA quantitation. Biotechniques. 2000 Aug;29(2):332-7. doi: 10.2144/00292rv02. PMID: 10948434.Taylor...Table 1: Features of various qPCR chemistries. Adapted from Wong, 2018   Primers Design of primers...

...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. Kleinstiver, Benjamin P., et al. "Genome-wide specificities...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell. 2015 Sep 23. pii: S0092-8674... the simplest CRISPR systems and the easiest to adapt to genome editing, but the introduction of type ...positions are indicated below each graph. Figure adapted from Kleinstiver et al., 2016. Until recently...

...testing your AAV.  Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8, and 9 in two different ...) your experimental conditions and measurements; (2) the cells you’re targeting; (3) how much expression... Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLOS ONE, 8(9),... Comparison of four promoters. Gene Therapy, 28(1–2), 56–74. https://doi.org/10.1038/s41434-020-0169-1...analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral...Brans, M. A. D., Luijendijk, M. C., Garner, K. M., & Adan, R. A. H. (2010). Optimization of Adeno-Associated...

... the two DNA strands and releasing topoisomerase (2).  These days commercially available TOPO kits provide...Taq polymerase using your favorite PCR protocol.  2. Set Up TOPO Cloning Reaction: Mix together the PCR...PubMed PMID: 1658796. PubMed Central PMCID: PMC52876. 2. Novel approach to molecular cloning and polynucleotide...however, the TOPO cloning technique has also be adapted for blunt end cloning. So how does TOPO cloning...

...phages but to also destroy the phage upon re-exposure (2, 3). More specifically, specialized Cas proteins snip..., accessed 25 November 2013. Author: James Atmos (2). Many CRISPR/Cas moieties carry out these functions...originally utilized CRISPR/Cas as a powerful and adaptive defense strategy against life-threatening viruses...scientists to hypothesize that prokaryotes developed an adaptive immune system that utilized various cas genes ... of anti-CRISPR genes in phages The CRISPR/Cas adaptive immune system seems like the winning ticket to...

...is unique compared to the rest of the genome and (2) the sequence is located just upstream of a Protospacer...pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9 homologs derived from species other...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi....applications. The first non-Cas9 CRISPR protein adapted for genome engineering was Cas12a (formerly Cpf1...targeting flexibility. Cas13a (formerly C2c2) has been adapted for targeted RNA cleavage in mammalian cells. The...Replacement) system fuses Cas13b to RNA deaminase ADAR2 to create a specific RNA editor. Cas13 enzymes are...

...the absence of genome editing (Fig. 2 and 3) (5, 3). Figure 2: Gene activation with nuclease-competent...shown to exhibit more limited activity than SpCas9 (2). In many cases this is due both to lower relative...PMID: 24076762. PubMed Central PMCID: PMC3844869. 2. Chari, Raj, et al. "Unraveling CRISPR-Cas9 genome...interacts with a ≤15nt gRNA that contains an RNA adapter capable of recruiting a transcriptional activator...

... were depositing and requesting each year (Figure 2). We did a similar analysis with CRISPR pooled libraries...different categories (Figure 2C). Note that in Figure 2, the numbers on the X axis refer to distribution and...rather than a cumulative total.    Figure 2: Addgene CRISPR deposit and distribution by function...rapid adoption of new tools, illustrated in Figure 2, and the longevity of tools seen in Figure 3, indicates...our requestors. However, we can again see quick adaptation and steady use of all types of libraries, once...

... Figure 2: Ironing boards. Good as a WFH desk, and as a video control center.   Cycle 2: Zooming ...ironing board, giving it the perfect height (Fig. 2). We recorded video using a simple, external video... Figure 1: The Design-Build-Test-Learn cycle adapted for video making.   Cycle One: The Ironing ...on-camera presentations and interviews. But how can I adapt this setup for the lab, so we can get back to producing...shoot. I also added a teleprompter to the mix, adapting the setup to run some remote interviews, so the...

...handled at BSL-1, while lentivirus requires a BSL-2 setup. This requires a less involved setup when working...infection rates in your experiment. Figure 2: Visual representation of common AAV serotypes used..., 10(1). https://doi.org/10.1038/s41467-019-12449-2 Pupo, A., Fernández, A., Low, S. H., François, A.,...References and Resources References Braun, C. J., Adames, A. C., Saur, D., & Rad, R. (2022). Tutorial: design...Teyssier, N., Pan, A., Danner-Bocks, S., Sanghal, T., Yadanar, L., Tian, R., Ma, K., Palop, J. J., & Kampmann...

...therapy had fallen - plummeted really -  from grace 2 years prior with the death of Jesse Gelsinger in an... the whole field to a grinding stop and only now, 2 decades later, is it finally back in the limelight...the other hand, have broad tropism in the eye. AAV-2, the first identified serotype and the one used in...reading 1. FDA approves gene therapy for blindness 2. Gene Therapy emerges from disgrace to be the next...efficiently transduce various types of retinal neurons. Nowadays a toolbox of naturally occurring and engineered...