...University of Manitoba due to their Biosafety Level-2 or 2+. Please Note: Despite the fact that AAVs themselves...Location (1): What tissue? Location (2): Where in the tissue/which cell type? Location (3): Traveling from...in very specific cell populations. Find Ready-to-Use Retrograde AAV Preps at Addgene 2) Comparison of genetically...long and stable expression (up to 9 months in mice [2]). Low Immune Response: Compared to other viral vectors...may be sufficient to overcome the transport issues. 2. Failure to plan (for storage) Is planning to fail...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 (...

...fluorescent state make them useful for 2-color imaging with green PA-FPs. Table 2: Properties of Selected Reversible...PA-FPs come in 2 flavours- monomeric and tetrameric. Tetrameric PA-FPS are better for whole cell imaging and... of proteins and cells." Science 297.5588 (2002): 1873-1877. PubMed PMID:12228718. 2.Subach, Fedor V.,... conventional FP imaging. PA-FPs are divided into 2 main categories - those with irreversible and reversible... forms of the chromophore which contribute to the 2 peaks of wtGFP’s excitation spectrum- a major 397 ... / N99K / S148L / I165V / Q167P / L169V / I203R) (2) and PA-mRFP1(S146H / I161V / I197H) (3) were derived...with two distinct wavelengths of light (see Table 2). The best known reversible PA-FP is the naturally...

... (details in [2]) and can be used to quantify rates of mutation in a bulk or single cell-derived genomic...resulting from non-homologous end joining (NHEJ) (2-5), depending on your desired genome edit. Since exploitation... have been reported for mismatch nuclease assays (2, 6, 7). Ability to distinguish between mono-allelic...assays, in addition to testing their performance (2). Remember that although digital PCR is a great screening...PMID: 24509632. PubMed Central PMCID: PMC4063274. 2. Findlay, S. D., Vincent, K. M., Berman, J. R. & Postovit... successfully analyzed gDNA from a few thousand cells from single wells of a 96-well plate without prior... that they are easily able to distinguish single-cell derived samples with mono-allelic mutations versus...

...Diverse Class 2 CRISPR-Cas Systems. Molecular Cell 60:385–397 . https://doi.org/10.1016/j.molcel.2015.10.008...Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi.org/10.1016/j.cell.2015.09.038 Additional... can be used to diagnose the presence of SARS-CoV-2 RNA, but inadequate access to reagents and equipment...named this system SHERLOCKv2 for SHERLOCK version 2. Here are some of the improvements: SHERLOCKv2 uses... a protocol for using SHERLOCK to detect SARS-CoV-2 RNA. The test is started using RNA purified from patient...recently adapted the DETECTR platform to detect SARS-CoV-2 using a lateral flow strip format. They published ...CY (2020) CRISPR–Cas12-based detection of SARS-CoV-2. Nature Biotechnology. https://doi.org/10.1038/s41587...

... of 2 components: 1) an SB transposase, the enzyme required for catalysis of transposition; and 2) a transposon...stable transgenic cell lines. Biotechnol J. 2015 Feb 4. doi: 10.1002/biot.201400821. PubMed. 2. Mates L, et... gene (red) for expressing the transposase enzyme.2.) The transposase enzyme is expressed (red stars) ...to infect and replicate in host cells, but they are susceptible to cells’ defense mechanisms. The use of... transposon; the plasmid is then degraded by the cell.4.) The transposase creates a double strand break...improve its gene insertion efficiency in primary cells. In 2009, Zsuzsanna Izsvak’s lab screened many SB...tranposase variants for hyperactivity in mammalian cells. Their efforts resulted in a new and improved SB...

...Gordon Fishell 83899-AAV1 pAAV-mDlx-GCaMP6f-Fishell-2 GCaMP6f expression in forebrain GABA-ergic interneurons...Gordon Fishell 83899-AAV9 pAAV-mDlx-GCaMP6f-Fishell-2 GCaMP6f expression in forebrain GABA-ergic interneurons...Gordon Fishell 83899-AAVrg pAAV-mDlx-GCaMP6f-Fishell-2 GCaMP6f expression in forebrain GABA-ergic interneurons...real-time interrogation and monitoring of brain cells and circuits during behavior. Long desired by researchers...A somatic channelrhodopsin, which enables single cell, single millisecond resolution optogenetics. Human...A somatic channelrhodopsin, which enables single cell, single millisecond resolution optogenetics. Cre-dependent...genetically encoded dopamine sensor RdLight1 in mammalian cells. Lin Tian 125708-AAV9 pAAV-hSynapsin1-RdLight1 Expresses...

...TUs. Level 2: Assembly of multiple Level 1 TUs into multigene constructs. Figure 2: The three ...can be queried by testing any number in the Level 2 assembly. These pathways can require a lot of optimization... of multigene constructs. PLoS One. 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765.  Hahn,... (2020). https://doi.org/10.1186/s12870-020-02388-2 Resources on Addgene.org Addgene’s Modular Cloning...complex cellular behaviors requires making complex manipulations to cells. For example: in a cell there ... yield a cellular phenotype, translational output, etc., which can support a larger cellular engineering...TU, or even an individual part, will behave in a cell. For unpredictable cases, MoClo provides two answers...

...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...

...expression and needing to work with a transfectable cell line. 2) They require larger expression vectors which...Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell,163(3), 759-771. PubMed PMID: 26422227...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... of 4 crRNAs in vitro and when expressed in 293 cells. This allows for a single promoter to drive expression... were used.  With transfection, 6.4% of HEK293T cells had edits at 4 of 4 targets when an array of 4 crRNAs...

...: PMC3337962. 8. Zhao S, et al. Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic ...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...

... H. J. (2009). Rats! Disease Models & Mechanisms, 2(5–6), 206–210. https://doi.org/10.1242/dmm.002733 ...are often used to study vertebrate embryology and cell development. One major resource for frog research...Organism to Study and Model Human Genetic Disease. Cells Tissues Organs, 205(5–6), 303–313. https://doi.org...

...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...

...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 ...

...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,...

...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...

... 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...

...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...

...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...