...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...

...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...

...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...

...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...

...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...

...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 ...

...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...

...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...

...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...

...nondividing cells and have an ~8kb packaging capacity. With this packaging capacity, reprogramming factors can...other delivery methods. Retrovirus has a cloning capacity of ~8 kb, so either a single polycistronic vector...or multiple single-gene vectors can be used to package reprogramming factors. An important milestone of...and nondividing cells and have a larger cloning capacity (8-10 kb) than other retroviral vectors. This ...a vector and chromosomal DNA. PB has a cloning capacity of ~9-14 kb. This system is transfected into cells...simple and fast to evaluate. iPSCs should form compact colonies that are comprised of cells with a large...differentiated in vitro to form embryoid bodies which are compact balls of loosely organized tissues that express...

...system is that it is too large to be effectively packaged into AAV particles for delivery into mammalian... much smaller and would be ideal for such AAV packaging, but their activity in mammalian systems has been... based systems, making it well suited for AAV packaging. Most importantly, this CasMINI system was shown...CasMINI plasmids Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs Yong-Sam Kim...result is a highly efficient Cas system with very compact genome editors and a gRNA down-sized by almost ...specifically binds its own mRNA to facilitate its packaging into virus-like particles. Feng Zhang’s lab adapted...the native PEG10 RNA sequences to instead drive packaging of cargo sequences in the capsid, in an approach...

... across regions, with Europe most impacted and China least impacted. The inferred weekend effect is, in...date range is, they could be the most impacted or least impacted. These results highlight the instability... last six months, COVID-19 has had a tremendous impact on our world -- as of June 4, 2020, COVID-19 has...unique PIs/labs placing orders per day) on dates impacted by each effect. The inferred parameters suggest...methods for fundamental scientific discovery and high-impact applications.       Alvin Hsu is a graduate student...next-generation therapeutics. Prime editing, base editing, PACE, and DNA-templated synthesis are four examples of...