...specific control of activity. Read More CRISPR Class 2 C lustered R egularly I nterspaced S hort P alindromic...receptors to selectively control and investigate cell signaling pathways. Receptors are remotely controlled...and genetic engineering to measure and manipulate cells (frequently neurons) and their governing biomolecular...detect, measure, and control molecular signals and cells in order to understand their functions. Read More...

...specific control of activity. Read More CRISPR Class 2 C lustered R egularly I nterspaced S hort P alindromic...receptors to selectively control and investigate cell signaling pathways. Receptors are remotely controlled...and genetic engineering to measure and manipulate cells (frequently neurons) and their governing biomolecular...detect, measure, and control molecular signals and cells in order to understand their functions. Read More...

...MO, Trono D, Aebischer P. Nat Methods. 2006. Feb;3(2):109-16. PubMed . In vivo RNA interference demonstrates...functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Rubinson...gene silencing mechanism. As a tool in mammalian cell systems, silencing is achieved through the delivery...expression; Designed for inducing RNAi in a wide range of cell types, tissues and organisms Luk Parijs 11619 pLB...Hacohen N, Hahn WC, Lander ES, Sabatini DM, Root DE. Cell. 2006. 124:1283-1298. PubMed . A versatile tool ...Lentivirus-delivered stable gene silencing by RNAi in primary cells. Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H,...

...Optogenetic control of intracellular signaling pathways. Trends in Biotechnology, 33(2), 92–100. https://doi.org...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 ...

...identification.   Fig. 2: Gel image of Plasmid 61564: pBAMD1-2 (~4.7 kb) digested with SpeI and...to the formation of plasmid multimers. Cell. 1976 Dec;9(4 PT 2):707-16. doi: 10.1016/0092-8674(76)90134... at ~3.6 kb and ~1.1 kb (lane 3). Uncut DNA (lane 2) has a band at <3 kb, which corresponds to the supercoiled...dimers in Escherichia coli K-12. Plasmid. 1989 Mar;21(2):138-41. doi: 10.1016/0147-619x(89)90057-7. PMID: ...Plasmids and Chromosomes. Microbiol Spectr. 2014 Oct;2(5). doi: 10.1128/microbiolspec.PLAS-0025-2014. PMID...Behavior of Plasmid DNA. Microbiol Spectr. 2015 Apr;3(2):10.1128/microbiolspec.PLAS-0036-2014. doi: 10.1128...at lower copy numbers within bacterial cells, and bacterial cells with multimers grow more slowly compared...

...Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme...BclI-SwaI none S. pyogenes URA3 Wyrick pCRISPomyces-2 61737 Bacteria BbsI yes, cut S. pyogenes Apramycin... S. pyogenes Zhang gRNA Cloning Vector Bbs I ver. 2 85586 Mammalian BbsI None S. pyogenes H Fujii pX601...Cas protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both...Alternatively, gRNA-only plasmids can be used in cells or organisms in which Cas9 has been integrated into...

... we identified in step 2. The result is a heterogenous population of stem cells, usually housed in a single...: 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...gRNA sequences without off-target effects for the 2+ million DHS sites. See below for further details.... for stem cells, or metastasized tumor cells, or alzheimers-affected neurons, or [your cell line of interest...critical to stem cell survival. Knowing which genes influence the survival of our cell culture is the foundation...

...question: C2c1, C2c2 and C2c3 (short for Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1,...characterization of diverse class 2 CRISPR-Cas systems." Molecular cell 60.3 (2015): 385-397. PubMed PMID...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...thought to activate programmed cell death or a dormant state for bacterial cells that have been infected with...bacterial cells, LwaCas13a does not display nonspecific RNA cleavage activity in eukaryotic cells. They arrived...target RNAs, although its usefulness in bacterial cells will likely be limited by the propensity of Cas13a...

...Tethering partner Subcellular localization 1 TagBFP 399nm/ 456nm histone 2B (H2B) Chromatin 2 Cerulean 433nm...based (chaining-based) cloning. The modules (Figure 2) are contained in individual plasmids and are each...between various cloning methods are listed in Table 2. For example,  chaining-based cloning methods require...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...protein localization, as well as cellular processes like gene expression and cell migration (Sladitschek and... MXS-chaining were used to label subcellular structures in live cells. Four individual expression constructs...

...PubMed Central PMCID: PMC3640491. 2. Grimm, D., et al. "Titration of AAV-2 particles via a novel capsid ELISA...report the physical titer of a viral solution (Figure 2). Because physical titers are used for dosing purposes...mean and how they can be compared.   Figure 2: Label that comes on Addgene’s AAV aliquots. Our titer...of genomes can limit production of recombinant AAV-2." Gene therapy 6.7 (1999). PubMed PMID: 10455443. ...Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material." Human gene therapy 21.10...particles that can transduce cells. Infectious titers are typically quantified by cell transduction assays. Wild-type...

...kinetics which are illustrated in figures 2 and 3.  Figure 2: Glutamate titration curves for the different...signals upon sensing glutamate using both 1-photon and 2-photon imaging.   Figure 1: 1P and 2P excitation...adapted from Aggarwal et al…, Supplementary Figure 2.   Improved trafficking and localization through... glutamate release from neurons and other brain cells in vivo. Glutamate plays a variety of roles in synaptic...sensor to the membrane and displays it on the extracellular side. In addition to the original PDGFR TMD,...promoter, FLEXed for expression in Cre-expressing cells hSynap: Human Synapsin-1 promoter. Good for neuronal...promoter, FLEXed for expression in Cre-expressing cells. hSynap-FLP: Synapsin-1 promoter, FLP-dependent ...

...panel (Table 2) for a hypothetical experiment in the lab. Let's say I am culturing HEK 293T cells in vitro...weak. Table 2: A simple staining panel to detect live, transduced, and CD45-expressing cells. Marker.... 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 561nm...relatively stronger—or brighter—signal.   Figure 2: Excitation and emission spectra for PE. The excitation... (eGFP expression).     The first stain 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...

...8661 p4455 FLAG-hPLIC-2 UBQLN2 Flag CMV ALS Peter Howley 8662 p4456 FLAG-hPLIC-2 NTF UBQLN2 Flag CMV ALS...196208 GB1-A11(2-196)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196209 GB1-A11(2-196, G38R)-Strep...196210 GB1-A11(2-196, D40G)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196211 GB1-A11(2-196, G175R...196212 GB1-A11(2-196, G189E)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196213 GB1-A11(2-52)-Strep ...TLS 1: hTLS.pBSKS(+) FUS T7 ALS David Ron 21828 TLS 2: hTLS.pCDNA1 FUS CMV ALS David Ron 21829 TLS 3: TLS.pRSET.C...ApoE Alzheimer's Sohail Tavazoie 51436 pGL3Basic-ME.2/ApoEpromoter APOE ApoE Alzheimer's Sohail Tavazoie...T7 ALS Nicolas Fawzi 127195 RP1B FUS 1-163 QQ4xSS #2 FUS His T7 ALS Nicolas Fawzi 127196 pJ411 FUS 1-163...

...AAV2 vectors in the mouse retina. Mol Ther . Feb;19(2):293-301. PMID: 21045809 Other citations include: ...adipose tissue-derived mesenchymal stem cells into pluripotent stem cells by a mutant adeno-associated viral...serotype also demonstrates enhanced transduction of cells in culture for ex-vivo conditioning. The AAV2(trpYF...displays enhanced transduction of hematopoietic stem cells and microglia. The AAV6(dbY-F+T-V) serotype was ...AAV-mediated transduction and targeting of retinal bipolar cells with improved mGluR6 promoters in rodents and primates...human gene therapy of glaucoma and their reversed cell entry characteristics. Gene Ther . 2020 Apr;27(3...delivery of a novel AAV vector targets ON bipolar cells and restores visual function in a mouse model of...

...Addgene. 2. Ma, Hanhui, et al. “Multicolor CRISPR labeling of chromosomal loci in human cells.” Proc. ...two genes over time, at a range of distances from 2-75 Mbp. Labeling multiple, intrachromosomal loci Building...successfully resolved loci with physical map distances of 2 and 75 Mbp, with the calculated fluorescent distances...established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that they could evaluate the...Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System.” Cell 155 (2013): 1379-1491. Pubmed...localization and movement of this locus in live cells! This technique was developed in the lab of Addgene...et al. to track telomere dynamics throughout the cell cycle. To optimize the signal to noise ratio of ...

...Currently available from the Kim lab are 2 presynaptic and 2 postsynaptic targeting mGRASP plasmids. Additionally... Robinson et al., Dev Cell 2010 Feb 16;18(2):324-31. Cheeseman et al., J Cell Sci 2013 May 1;126(Pt ... 2µg/ml of Doxycycline for 2 hours, and imaged on the Keyence BZ-9000 fluorescence microscope 8 hours ...promotes the formation of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated... of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly...1 modules can be similarly assembled into a Level 2 vector, thus forming a functional genetic circuit....circuit. Flexibility has been built into the Level 2 vectors to allow for additional iterations of Level 1 assembly...

...purify IVT sgRNA in the lab. Figure 2: Cas9 RNP complexed with 2-part cr:tracrRNA tags genes more effectively...is then transfected into stem cells. Cells are allowed to recover for 2-5 days and then analyzed for tagging...transfect and let cells recover for 2-5 days.Note: We use the Nucleofection method for delivery of csRNP... knock-in efficiency (<2% positive by ICC for the tag) or do not see any cells positive for tag insertion...stem cells. Development 144: 635-648. PubMed PMID: 28096221. PubMed Central PMCID: PMC5312033. 2. Dewari...IVT sgRNA) approach (Dewari et al. 2018 and Figure 2). Not only is the cr:tracrRNA approach highly efficient...transfected cells, we count the number of tag-positive cells (tag ICC) as a percentage of total cells in the...

...either SARS-CoV-2, Adenovirus type 41 (AdV-41), or Adenovirus-Associated Virus type 2 (AAV2) – or some... was either 1) a physiological effect of SARS-CoV-2 toward destabilizing one’s natural immune response...response to Adenovirus (and/or to its partner, AAV) or 2) a sociological effect of the COVID-19 lockdowns causing...… Chiu, C. Y. (2023). Adeno-associated virus type 2 in US children with acute severe hepatitis. Nature... AdVs is dependent on modulation of the host cell’s cell cycle; inhibition of the innate immune system... in the producer cells where there is E1 and E3 gene complementation. In target cells that are infected...hexon), as well as other intracellularly-exposed proteins and downstream cellular events, all recruit strong...

...plasmid, or a 2-plasmid system in which Cas9 must be delivered separately.  Mutant cells are enriched ...targeting a specific gene within the genome (see Fig. 2). Individual gRNAs are designed in silico using publicly...mouse, human, fly, E. coli, and T. gondii genes.  2) What genetic modification are you trying to make?...CRISPR libraries is a multi-step process (see Fig. 2). In most cases, CRISPR libraries are provided at ...heterogeneous population of mutant cells, with each cell or set of cells containing a mutation in a different...fluorescence-based cell sorting and screened for a particular phenotype. For example, mutant cells can be used...lead to skewed results. Selecting a cell type - Theoretically, any cell type can be used in a CRISPR screen...

..., AAV2/8 has a type 2 ITR and a type 8 serotype. Almost all vectors contain type 2 ITRs, which comes from...terminally differentiated, non-dividing cells.   Figure 2: DNA rescue and transduction of a conventional...quantitative PCR-based method for the titration of AAV-2 vector stocks."Molecular Therapy 6.2 (2002): 272. ...Time to expression AAVs have to infect cells, hijack host cell machinery, and synthesize a second strand...that can be used to express genes of interest in cell and animal models. AAVs are non-pathogenic, do not...be specifically targeted to a number of different cell types even when systemically administered in an ...ranging from transient gene expression in a particular cell type to CRISPR genome editing, optogenetics, and...