...581 95 4.7 1 hr Tandem-dimer pCSCMV:tdTomato - Mammalian Expression pFA6a-link-tdTomato-SpHis5 - Yeast Expression...Expression mEGFP 488 507 34 6 Monomer (A206K) pmEGFP-1 - Mammalian Expression mEGFP-N1 - Mammalian Expression...-pBAD - Bacterial Expression mRFP1 584 607 13 4.5 1 hr Monomer pcDNA3-mRFP - Mammalian Expression pMXs-mRFP1... of Biomedical Optics, Feburary 2005, Vol. 10 No. 1, p. 14003 Chu et al. : Nature Methods, May 2014 Vol...Expression mT-sapphire 399 511 Monomer (A206K) mT-Sapphire-N1 - Mammalian Expression mT-Sapphire-C1 - Mammalian...Structure Plasmids T-sapphire 399 511 26 4.9 1.3 hr Prone to dimerization pFA6a-link-yETSapphire-CaUra3 - Yeast...cysteine-free SGFP2) ZsGreen 493 505 39 Tetramer pHIV-Zsgreen - Mammalian Expression (this is a bicistronic...

... most popular genome engineering approach. Figure 1: Overview of the basic CRISPR mechanism Engineered...from Staphylococcus aureus ) has a coding sequence ~1 kb shorter than SpCas9 while retaining the same basic...lentiviral transfer vectors (Figure 8B). Libraries come in 1-vector systems, in which Cas9 is included in the gRNA-containing...efficiency and fidelity. Nature Communications , 13 (1), 1425. PMID: 35301321 Edraki, A., Mir, A., Ibraheim...interrogation by SpRY-Cas9. Nature Communications , 15 (1), 3663. PMID: 38688943 Hsu, P. D., Scott, D. A., Weinstein...increase its specificity. Nature Communications , 9 (1). PMID: 30082838 Maddalo, D., Manchado, E., Concepcion...CRISPR-Cas9 with Bacteriophage Proteins. Cell , 168 (1–2), 150-158.e10. PMID: 28041849 Sakuma, T., Nishikawa...

...researchers use ssDNA or dsDNA repair templates with 1. homology to the DNA flanking the DSB and 2. a specific...gRNA Vectors gRNAs By Species Mammalian Bacteria Drosophila Plant C. elegans Yeast Zebrafish Xenopus CRISPR...for expression in mammalian systems, bacteria, Drosophila, plants, C. elegans, yeast, zebrafish, and Xenopus...ID Plasmid Gene/Insert Promoter PI Publication Drosophila ID Plasmid Gene/Insert Promoter Selectable Marker...

...some mammalian systems have been developed. Figure 1: Overview of the basic CAST mechanism. Created with...gRNA Vectors gRNAs By Species Mammalian Bacteria Drosophila Plant C. elegans Yeast Zebrafish Xenopus CRISPR...

...proteins in budding yeast. Sci Rep. 2019 Feb 19;9(1):2234. doi: 10.1038/s41598-019-38913-z. PubMed 30783202...Lee et al., 2013), and are available with URA3, SpHIS5 and KANMX markers. The FP together with the protothropic...

...available as a substrate for another round. Figure 1: Overview of prime editing. Created with BioRender.com...OsU3 pegRNA No Yiping Qi 149545 pCFD3-NS Drosophila Drosophila U6:3 pegRNA BbsI No Vermilion Norbert Perrimon...Perrimon 149546 pCFD5-NS Drosophila Drosophila U6:3 pegRNA + nicking sgRNA BbsI No Vermilion Norbert Perrimon...gRNA Vectors gRNAs By Species Mammalian Bacteria Drosophila Plant C. elegans Yeast Zebrafish Xenopus CRISPR...expression in mammalian systems, bacteria, plants, and Drosophila. Mammalian ID Plasmid Gene/Insert Promoter Selectable...Insert Promoter Selectable Marker PI Publication Drosophila ID Plasmid Gene/Insert Promoter Selectable Marker...

... Caltech and can help guide your decision. Figure 1: Decision tree for choosing your AAV capsid. Your ...tdTomato Control Boyden 83900 pAAV-mDlx-GFP-Fishell-1 Dlx GFP Control Fishell 99130 pAAV-mDlx-NLS-mRuby2...for the AAV Decision Tree The AAV decision tree graphic above helps you choose the best capsid for your...

...    Figure 1: A 1 mg/mL control is diluted in a series ranging from 1:2 to 1:32. Each individual...example, Figure 1 depicts a 2-fold dilution series. Each step of the series dilutes by 1:2 for a series...series that ranges from 1:2–1:32. The specific dilution series used will depend on the expected concentration... points fit the trendline. R2 ranges from 0 to 1 with 1 being a perfect fit and 0 indicating that there...individual step of the series is consistent at 1:2. When preparing a dilution series, use a new pipette tip for...absorbance is plotted on the y-axis. With the help of graphing software, you then generate a trendline for the...the quality of a standard curve scientists use graphing software to calculate the data’s coefficient of...

...Cells? Yes Yes Longevity in Serum?* No Yes Table 1. Comparing SpCas9 and GeoCas9. *For in vitro Cas9 ...GeoCas9 is active at high temperatures Figure 1: Thermostability of SpCas9 vs GeoCas9. SpCas9 or GeoCas9...from 25-80 °C. Activity is expressed as Kcleave (min-1). SpCas9 has maximum activity from 35-45 °C, while...outperformed SpCas9 at high temperatures. See Figure 1 for a summary of the experimental results. GeoCas9...from the thermophilic bacteria Geobacillus stearothermophilus (GeoCas9). G. stearothermophilus grows at...cells, yeast, drosophila, plants, worms, zebrafish, frogs, some bacteria; but not thermophilic (high heat...for facultative thermophiles (high OR medium heat loving), but not obligate thermophiles. However, the ...

...approximately 1 in 5000 males. DMD is caused primarily by frameshift mutations in dystrophin, a protein ... applicable to 80% of DMD patients. Figure 1: Exon skipping strategies for Duchenne muscular dystrophy...needed. First, CRISPR delivery must be optimized to: 1. Reach a high percentage of muscle cells throughout...also be closer than we might expect.   References 1. Kaiser J. CRISPR helps heal mice with muscular dystrophy...neurogenetic diseases. Nucleic Acid Ther. 2014 Feb;24(1):69-86. PubMed PMID: 24506781. PubMed Central PMCID...);186-91. doi: 10.1038/nature14299. Epub 2015 Apr 1. PubMed PMID: 25830891. PubMed Central PMCID: PMC4393360...for proper muscle function. Without functioning dystrophin, an individual experiences progressive muscle...

...building it! Table 1 has a list of Fragmid destination vectors currently available. Table 1: Fragmid destination...components on the Fragmid website.   Figure 1: (Top) Schematic overview and timeline of the Golden... CRISPR vector the Fragmid way!    Figure 1: Selecting your vector type on the Fragmid website...components available will depend on your answers in step 1, so you’ll only be able to select components that ...attR sequences for genome integration by phiC31) Drosophilia Plasmid pRDB_052 (for stable fly cell... pRDA_791 (contains only orio and ampR) Drosophilia Plasmid pRDB_051 (for stable fly cell lines...integration by spontaneous insertion)   Drosophilia Plasmid pRDB_053 (for transgenic flies; gypsy...

...64325 3xFLAG-dCas9/p-bacteria 3xFLAG-dCas9 pLtetO-1 Fujii Efficient isolation of specific genomic regions...gRNA Vectors gRNAs By Species Mammalian Bacteria Drosophila Plant C. elegans Yeast Zebrafish Xenopus CRISPR...

...Table 1 has a detailed list of the bacterial species these plasmids are compatible with.  Table 1: Non-...reduced metabolic load (Meyer 2009). Figure 1: Idealized examples of a narrow host range plasmid...for DNA cloning in Gram-negative bacteria. Gene, 70(1), 191–197. https://doi.org/10.1016/0378-1119(88)90117... N.J.), 765, 327–343. https://doi.org/10.1007/978-1-61779-197-0_19  Meyer, R. (2009). Replication and ...Gluconacetobacter xylinus, Haemophilus influenzae, Hypomycrobium X, Legionella pneumophila, Methylophilus methyltrophus...Escherichia coli, Klebsiella spp., Legionella pneumophila, Methylophilus methylotrophus, Myxococcus virescens,... A. viscosus, Aerobacter aerogenes, Aeromonas hydrophila, Agrobacterium tumefaciens, Alcaligenes eutrophus...

...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems...archaea, though Class 1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible...optimal plasmids to harness the power of Class 1 systems. Class 1 CRISPR systems can be further broken down...types, 33 subtypes, and 17 variants. Figure 1: Overview of the CRISPR classification hierarchy. ...effector complex after binding to a target. Class 1 is the most abundant of the two, comprising about ...incredible abundance, Class 1 CRISPR systems are less popular in the lab. This can be attributed to the multi-Cas...Type I CRISPR systems make up the majority of Class 1, and are the most common type of CRISPR overall. Type...

...of publication. Table 1 provides an example calculation of the weights. Table 1: Journal Weighting Example...amassed nearly 2,400 citations in its first four years (1). In addition to publishing in Science, Professor ...papers without plasmids deposited with Addgene (Figure 1). Even aggregated this much, you can see the 2013...(F) Weight (=F/E) (G) Weighted Citations (=C*G) 1 2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010...website at: http://www.neil-t.com/ .   References 1. Cong, Le, et al. "Multiplex genome engineering using...highly cited, we went to one of the largest bibliographic databases available, Web of Science. For each... publication, Web of Science provides basic bibliographic information and uses its vast network of articles...

...PDF 2.3 MB Zhang GeCKO pooled library amplification 1 vector system: lentiCRISPR v2 2 vector system: lentiCas9...putative target sites for S. pyogenes Cas9, S. thermophilus Cas9, or Cpf from your input sequence or list...checks for off-target binding. Currently supports: Drosophila, Arabidopsis, zebrafish, C. elegans, mouse, human...tropicalis and X. laevis), zebrafish, sea squirt, Drosophila, C. elegans, Arabidopsis, rice, sorghum, silkworm...checks for off-target binding. Currently supports: Drosophila, Arabidopsis, zebrafish, C. elegans , mouse, ...off-target binding and can work for S. pyogenes, S. thermophilus or N. meningitidis Cas9 PAMs. Currently supports...

...BD, Clevers H. Cell. 2010 Oct 1;143(1):134-44. PubMed. Drosophila Brainbow: a recombinase-based fluorescence...the Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow...subsequent recombinations cannot occur. While the Brainbow-1 system employs Cre-mediated deletion, the design of...antibodies specific to each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained a “default” state; for...dendritic processes not previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained...study model organisms such as Drosophila and zebrafish. Brainbow in Drosophila has aided in the mapping of...tracing in other systems, such as zebrafish and Drosophila. Why Brainbow? Brainbow highlights the trend ...

...Hyg Chen 86196 pYPQ141-ZmUbi-RZ-As Maize ubiquitin 1 none As Cpf1 Qi 91715 pKEE401 yes, cut S. pyogenes...gRNA Vectors gRNAs By Species Mammalian Bacteria Drosophila Plant C. elegans Yeast Zebrafish Xenopus CRISPR...

...2 (CRY2) and calcium and integrin-binding protein 1 (CIB1) proteins. The goal of both groups was to create... the Sata lab is composed of two fusion proteins: 1) the genomic anchor - an inactive, dead Cas9 protein...combination. The optimized LACE system consisted of: 1) CIBN-dCas9-CIBN, where CIBN is the N-terminal fragment.... crassa) (Kawano et al., 2015). Nicknamed paCas9-1 and consisting of the fusion proteins N713-pMag and...fold-induction of Cas9 activity (16.4-fold). This paCas9-1 light-inducible system was able to recognize the same...flCas9). When triggered by blue light (470nm), paCas9-1 induced indel mutations via NHEJ (frequency of 20.5%...background activity of the system by modifying paCas9-1 using nMagC714 instead of nMagHigh1-C714, generating...

...which is also gaining a lot of new traction lately (1, 2), depends on knowledge of the structure(s) a given...RBPs successfully identified using this method. Step 1: Prepare a random fragment library of phage DNA This...first step involves digesting phage genomic DNA into 1-3 kb (approximately gene-sized) fragments so that ...in the bulk of resultant fragments falling between 1 and 3 kb results in optimal likelihood of capturing...solution-soaked filter paper (colony-side up) and leave for 1 h at room temperature. After this, it is helpful to...proteins expressed by your library. After blocking for 1 h at room temperature with gentle shaking, gently ... to incubate the membranes overnight in 500 mM to 1 M NaCl in order to enhance folding of the recombinant...