...with alternating spacers and repeats, was the original impetus for studying CRISPR. Spacers: Copies of foreign...sequences are called protospacers. Repeats: Bacterial DNA that appears between each spacer in the CRISPR array...the CRISPR array and include a spacer and a repeat sequence. The spacer sequence is the RNA guide, like...real mouthful of a phrase: Clustered Regularly Interspaced Short Palindromic Repeats. That contrast of simplicity...composed of identical repeat sequences and variable spacer sequences. The array is transcribed and processed... into crRNAs, each including one repeat and one spacer. In bacteria, these crRNAs are bound by Cas proteins... sequence base pairs with the tracrRNA, and the spacer sequence is used to target complementary DNA sequences...

...for expression of a ZFN targeted to a site with a spacer of 7 bps and harboring a “+” heterodimeric FokI...for expression of a ZFN targeted to a site with a spacer of 7 bps and harboring a “-” heterodimeric FokI...expression of a ZFN targeted to a site with a 5 or 6 bp spacer and “+” KKR heterodimeric FokI domain. ( Doyon ...expression of a ZFN targeted to a site with a 5 or 6 bp spacer and “-” ELD heterodimeric FokI domain. ( Doyon ...

...well, though. Viral packaging and entry The genetic cargo requires viral packaging to infiltrate and integrate...from the transfer plasmid. Viral packaging and entry The viral packaging proteins for AAV are rep and cap...Addgene blog Parts of the AAV packaging plasmid Overview of Lentiviral Packaging Plasmids Adenoviral production...multiple plasmids with many components are used to package lab grown viruses. It can be intimidating at first...transfer plasmid while the other plasmids with packaging or replication components are called helper plasmids...). These LTRs define the limit of what will be packaged. The 5’ LTR and 3’ LTR, respectively, act as RNA...nucleocapsid viral components needed for viral packaging, assembly, and display. Pol, the last component...

... Viral Vector Packaging Service AAV Molecular Tools Viral Vector Packaging Service: Molecular Tools..., Wells Don’t See What You’re Looking For? Our Packaged on Request service offers you even more options...Please note this does not guarantee viral vector packaging service, but lets us know what viruses would be...

... Viral Vector Packaging Service AAV Serotype Testing AAV Viral Vector Packaging Service: Serotype ...order it? Don’t See What You’re Looking For? Our Packaged on Request service offers you even more options...Please note this does not guarantee viral vector packaging service, but lets us know what viruses would be...

...have different packaging capacities depending on the virus. AAV has a packaging capacity of ~4.7 kb, while...containing your gene of interest plus two packaging plasmids (three packaging plasmids for 3rd generation lentivirus...Baculovirus transfer plasmids have a much larger packaging capacity — up to 38 kb! Generally, insect cell production...lentivirus production). The packaging plasmids are widely available, so you’ll only have to worry about cloning...while lentivirus has a capacity of 8–10 kb. Time Viral production in mammalian cells is relatively quick...construct, called a bacmid. Like AAV and lentiviral packaging plasmids, bacmids are readily available, so the...if needed # of plasmids Three or four Two Packaging limit ~4.7 kb (AAV); 8–10 kb (lenti) 38 kb Cells...

... Viral Vector Packaging Service AAV Recombinases Viral Vector Packaging Service: Recombinases ... Page . Don’t See What You’re Looking For? Our Packaged on Request service offers you even more options...Please note this does not guarantee viral vector packaging service, but lets us know what viruses would be...

...into an AAV vector is challenging due to its packaging capacity of AAV (~4.7 kb). While this approach has...previously packaged Cas9 and multiple gRNAs into separate AAV vectors, increasing overall packaging capacity...preferential infection of certain tissues. However, packaging Streptococcus pyogenes (SpCas9) and a gRNA together... the only CRISPR enzyme that’s small enough to package into AAV. At 984 amino acids in length, Cas9 from...Cas9 (NmeCas9) at 1,082 amino acids, can also be packaged into AAV. NmeCas9 has the added advantage that...respectively, the size of both is within AAV’s packaging limit. For CRISPR-based RNA editing, the REPAIR...approximately 4.1 kb in length, allowing them to be packaged in AAV.   Split AAV approaches  While finding...

... Viral Vector Packaging Service AAV Monosynaptic Neuronal Tracing Viral Vector Packaging Service: Tools...17329205 Don’t See What You’re Looking For? Our Packaged on Request service offers you even more options...Please note this does not guarantee viral vector packaging service, but lets us know what viruses would be...

... Viral Vector Packaging Service AAV Chemogenetics Viral Vector Packaging Service: Chemogenetics...Chemogenetics AAV Don’t See What You’re Looking For? Our Packaged on Request service offers you even more options...Please note this does not guarantee viral vector packaging service, but lets us know what viruses would be...

...high capacity AdV) are devoid of all viral coding sequences and retain only the ITRs and packaging signal... Ad vectors (helper-dependent (HD-Ad), or high-capacity (HC-Ad__ are devoid of viral genes and therefore...kb. Gutless Ad maintain only the ITRs and the packaging signal (Ψ), both of which are essential for assembly...such as HEK293 cells. 1st generation AdV cloning capacity is limited to 8.2 kb, and in vivo transgene expression...RCA) can be generated if the E1 gene from the packaging cell line is transferred into the AdV by recombination...Gen AdVs were designed to have increased cloning capacity, decreased  potential for the generation of RCA...2nd generation AdVs, thereby increasing cloning capacity to ~12 Kb. The ever popular pAdEasy system belongs...

...contaminating your transduction with the packaging cells. Packaging cell debris can be removed either by ... particles in a smaller volume. Always remove packaging cell debris before proceeding to ultracentrifugation...step (5 minutes at 300-500 g). After removing packaging cell debris, virus can be pelleted by ultracentrifugation...reduce the volume of cell culture medium on the packaging cells immediately following transfection. In other...fold (Stockschläder et al., 1999). 5. Check your packaging cells for infection If your transductions aren... virus is being generated by your transfected packaging cells. This check can be performed routinely and...disrupting the flow of your experiment. If your packaging cells have been successfully transfected and produced...

...Service picks up mail. Recycle plastic packaging Packaging is a huge contributor to the increase in ...lot of their plastic packaging (from Corning®, Falcon®, or Axygen® product packaging) for free! It has to...the lab could make a big difference in our global impact as scientists. Many scientists are already leading...toward responsibility in our lab’s environmental impact, but we can all do more to make research more environmentally...your address sticker off the box, remove the ice pack (it’s not recyclable), tape the box shut, and drop... to be from their company and it has to be packaging (no coated tissue culture plates, for example). Check...and companies have the potential to make a huge impact directly on waste, but also on culture. Encourage...

...details about our viral vector packaging service, visit our Viral Vector Packaging Service page . Pooled CRISPR...lentivirus available from Addgene's viral vector packaging service. Browse constructs currently available... Viral Vector Packaging Service Lentivirus Browse In-Stock Lentivirus Pooled CRISPR...

...host genome. AAVs are limited by their small packaging capacity. Adenovirus Plasmids | Guide Adenoviruses...type, linked above. Virus Expression Genome Packaging Capacity Virus Size (nm) Cells Infected Target Cell...

... Viral Vector Packaging Service AAV Controls Viral Vector Packaging Service: Control AAV Browse... Page . Don’t See What You’re Looking For? Our Packaged on Request service offers you even more options...Please note this does not guarantee viral vector packaging service, but lets us know what viruses would be...

...analyses needed for packaging AAV. AAV production Overview: The first step in packaging AAV is transfecting...had never worked with AAV, so naturally I had to package some virus for my first experiment. It was a bit...transfecting HEK293 cells with AAV packaging plasmids. The cells need three different plasmids to produce AAV...intermediate into the single-stranded genome that's then packaged into AAV’s protein shell, or capsid; 2) the pHelper...plasmid which encodes a transgene of interest that’s packaged into the virus. Two to five days after transfection...plasmids contain ITRs which are required for proper packaging of AAV, but also form secondary structures that...spreadsheet similar to this one to plan my AAV packaging PEI transfections. It saves me time and helps ...

... Viral Vector Packaging Service Penn Vector Core Transfer Viral Vector Packaging Service: Penn Vector...Optogenetics Scott Sternson AV-1-20071P 20071-AAV1 pACAGW-ChR2-Venus-AAV Optogenetics Karel Svoboda AV-1-...pAAV-hsyn-Jaws-KGC-GFP-ER2 Optogenetics Ed Boyden AV-9-20071P 20071-AAV9 pACAGW-ChR2-Venus-AAV Optogenetics Karel Svoboda AV-9-...-tdtomato Scott Sternson AV-5-20071P 20071-AAV5 pACAGW-ChR2-Venus-AAV Karel Svoboda AV-5-20949P 20949-...

...average only 2.8 kb in size and are easy to package in low-capacity vectors! These small, but mighty type VI-D...Cas9! Their small size makes then easy to package in low-capacity vectors like AAV, a popular vector due ...HEPN-independent. These enzymes also do not require a protospacer flanking sequence, so you can target virtually...either an unprocessed or processed gRNA array (22 nt spacer with 30 nt direct repeat) mediating potent knockdown...dementia-associated tau, delivering dCasRx and a 3-spacer array targeting the MAPT exon 10 splice acceptor...toolbox will be. It is, however, certain that the impact of CRISPR scientific sharing will continue to grow...Addgene. Yan, Winston X., et al. “Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively...

...think we are currently at in the CRISPR software space. The CRISPR research community hasn't had a chance...review process has been challenged with many closely spaced CRISPR software submissions. With few prior publications...GUI”. Trends: Online tools dominate the software space but generally rely on less powerful algorithms to...user in different formats. This can have a large impact on downstream results. Examples: “HTML”, “visual...agnostic tools are the better solution but are often packaged as offline tools requiring expertise in one programming...are important to define for the CRISPR software space. It is unlikely that you will find these terms anywhere... data dump of ALL strings in a genome using any IUPAC encoded pattern at any edit distance. It won’t, ...

...insert into a viral particle (the packaging signal Psi). Because the packaging plasmids lacked the psi signal...plasmid and the packaging plasmid caused the Psi sequence to be placed on the packaging plasmid forming...virus (MoMLV) and consisted of two plasmids: the packaging plasmid containing all the structural genes necessary...virus. Indeed, by testing several vectors and packaging cell line combinations, Miller et al. showed that...generated by the transfer of a retrovirus into the packaging cell line PA12 (containing the psi deletion). ...viral genes with the exception of the env gene (packaging plasmid), and 3) the vesicular stomatitis virus...293T cells, with your transfer plasmid and your packaging plasmids. For this, you can use the transfection...

...to using AAV is the limited packaging capacity. Lentiviruses have capacities of 8–12 kb, while AAV only...libraries available at Addgene. The large packaging capacity and stable expression of lentiviral vectors...Lentivirus has limited packaging options, with most lentiviral vectors being packaged using VSV-G. In contrast...contrast, AAV can be packaged in a variety of capsids. To date, at least 12 different capsids, or serotypes...that when using Cas9, you’re already almost out of space! Don’t let this get you down for too long, though...

...to the amount you are dispensing can negatively impact your experimental results. This protocol will guide...

...evolved optimizations for prime editing CHARM: A compact epigenetic silencer Recombinant antibodies for ...assembly of biotinylated SpyTag-SpyCatcher linkers and spacer proteins ending with a GFP nanobody (Figure 1)....determination. A) Schematic of workflow. B) Detail of spacer peptides (3HB: 11-nm 3-helix bundle; SAH: 60-nm...software (green circles), magnetic bead (*), and spacers are labeled; scale bar: 100 nm. D) 3D model of ...other targets. The modular system of linkers and spacers could enable many future variations using nanobodies... the PE components to be delivered in a single package. In a recent publication, the Liu lab showed that...doi.org/10.1038/s41551-024-01227-1   CHARM: A compact epigenetic silencer By Emily P. Bentley The Weissman...

...further! You can package your own lentiviral vector using an integrase-deficient packaging plasmid available... the integrase-deficient packaging plasmid instead of the standard packaging plasmid. For more details...integration of this portion into the host genome. The packaging plasmid contains Gag, Pol, Rev, and Tat genes....transcriptase), and IN (integrase). These viral packaging genes are required for the production of lentiviral...integrated into the host genome via the viral proteins packaged during production (read our blog post on the retroviral...integrated portion does not contain any of the viral packaging genes, the natural cycle of the lentiviral vector...details on how to package lentiviral vectors, check out Addgene’s Lentiviral Guide and our lentiviral production...

...Fisher 26402 pF141 pAcGFP1 SOD1WT SOD1 GFP CMV ALS Elizabeth Fisher 26403 pF142 pAcGFP1 SOD1 A4V SOD1 GFP...Fisher 26404 pF143 pAcGFP1 SOD1G37R SOD1 GFP CMV ALS Elizabeth Fisher 26405 pF144 pAcGFP1 SOD1G85R SOD1 GFP... GFP CMV ALS Elizabeth Fisher 26406 pF145 pAcGFP1 SOD1G93A SOD1 GFP CMV ALS Elizabeth Fisher 26407 pF146...Prnp_SAM2 PRNP U6 Dementia Walker Jackson 79259 pAc5.1B-lambdaN-HA-DmeIF4G-V5His6 EIF4G1 His, HA, V5 Ac5...IDR-mCherry TBP Parkinson's Denes Hnisz 146392 pAc5.1B-lambdaN-HA-DmeIF4G-trunc-V5His6_G EIF4G His, HA...HA, V5 Ac5 Parkinson's Elisa Izaurralde 146493 pAc5.1B-lambdaN-HA-DmeIF4Ga-Nterm_H EIF4G His, HA, V5 Ac5... Ac5 Parkinson's Elisa Izaurralde 146577 pAc5.1B-lambdaN-HA-DmeIF4Ga-dsRNAres_I EIF4G His, HA, V5 Ac5 ...

...new teaching space at Lab Central to learn all about it. LabCentral is a lab and office space shared by ...and making space for the techniques ourselves. As you probably have limited time and space in your own...by numerous emerging biotech companies. The space was modern with clean bold colors of orange and white...a few goodies which included an NEB drawstring backpack and a double wall insulated glass to take home...like workshops at Lab Central. Because of the fast pace of work in the Addgene lab, we don’t often get the...opportunity to explore new techniques in our own space. Workshops such as this give us a chance to learn...

...RNA provided that target is upstream (5') of a protospacer adjacent motif (PAM) Cas9 endonuclease binding... RNA and a 3-base pair sequence known as the Protospacer Adjacent Motif or PAM. In order for dsDNA to ... and the unique stretches of DNA between them “spacers” (see figure below). After years of research, we... each direct repeat, combined with its adjacent spacer, ultimately encodes a single crRNA. The direct ...pre-crRNA into mature crRNA and tracrRNA binding. The spacer regions, on the other hand, are the unique, foreign...tracrRNA forms the scaffold portion of a gRNA and the “spacer region” forms the target sequence.   Overview...transcribed into pre-crRNAs containing both the spacer region and the direct repeat region. B) RNase III...

... and infect specific cells. This protein shell packages the vector’s genetic material, and variations ...and can be selected as a serotype for Addgene’s Packaged on Request service. Some AAV1 tools are available...and can be selected as a serotype for Addgene’s Packaged on Request service. Some AAV2 tools are available...and can be selected as a serotype for Addgene’s Packaged on Request service. Some AAV5 tools are available...and can be selected as a serotype for Addgene’s Packaged on Request service. Some AAV8 tools are available..., and retinal cells (Issa et al., 2023). AAV9 packaging plasmids are available at Addgene. AAV9 vectors...vectors are not available through our Packaged on Request service, but some AAV9 tools are available as in-stock...

...Cas9 multiplexing in systems with limited packaging capacity. Still confused about the various tRNA expression... will be necessary to improve its multiplexing capacity. Multiplexing is key due to the natural redundancy...often necessary. Guide RNAs (gRNAs) are commonly packaged in 400-500 bp cassettes containing the RNA pol...100 bases) limit the number of gRNAs that can be packaged together in a single vector. In addition, the ...forms tRNA-gRNA and tRNA-gRNA-tRNA, with short 5’ spacer sequences preceding gRNAs, to test for gRNA cleavage...predicted with no addition of nucleotides to the 5’ spacers. The 3’ ends were modified slightly with either...once, and each <200 bp tRNA-gRNA takes up half the space of a standard pol III-gRNA, which will facilitate...