...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids Topics: CRISPR, Other CRISPR... where we highlight a few of the newest CRISPR plasmids available at Addgene. If you want to read more...will cover: Miniature CRISPR-Cas systems SEND plasmids Prime editing improvements Miniature CRISPR-Cas...scaffold gRNA and making strategic mutations in the DNA binding cavity of Cas12f. The CasMINI system is less...Read the paper in Molecular Cell Find the CasMINI plasmids Efficient CRISPR editing with a hypercompact ...paper in Nature Biotechnology Find the Cas12f-GE plasmids Programmed genome editing by a miniature CRISPR-Cas12f...cells using various delivery methods, including plasmid, ribonucleoprotein, and AAV.  Read the paper in...

... usage bias in Plasmids 101: Codon usage bias Read more about plasmids in our Plasmids 101 blog series...without altering the sequence of the protein. As DNA synthesis has become quite cost-effective it seems...seems easy for scientists to re-synthesize DNA with the most optimized sequence. Unfortunately, it is not...algorithms/resources (Athey et al., 2017) and many DNA synthesis companies, such as IDT, Genewiz and GenScript...available online to both determine and synthesize DNA sequences for optimal expression in your system of... (2000) Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic...Reference Read about genetic code expansion Browse our plasmid collection page  ...

...transforming both fusion plasmids, or mating two single haploid yeast fusion plasmid transformants into a ... in the cell, including protein-protein, protein-DNA and protein-RNA interactions. The 1980s saw a flurry...format of some transcriptional activators: that the DNA binding domain (DBD) and transcriptional activation...reporter system (e.g. the Gateway-compatible bait Y2H plasmid pEZY202  from Yu-Zhu Zhang’s lab) is an example...interaction detection methods (6). In order to bind DNA and activate a reporter gene in eukaryotic cells,...Keegan, L., Gill, G., and Ptashne, M. Separation of DNA binding from the transcription-activating function...eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell. 1985...

...you will also need to deliver a donor plasmid or single-stranded DNA oligonucleotide, so you will need to...editing? Do you want to deliver the components as DNA, or would mRNA or protein delivery be more suitable...transduction or injection as either protein, RNA, or DNA. Finally, once you have identified the best expression...generate stable cell lines. Packaging and Envelope plasmids provide the necessary components to make lentiviral...for in vivo viral delivery. Cas9 mRNA and gRNA Plasmids containing gRNA and Cas9 are used in in vitro ...efficiency of CRISPR delivery. There are a few plasmids at Addgene that have been published as CRISPR ...genome target sequence into EGFP reporter. Target DNA reporter system: Can evaluate component delivery ...

...repository for DNA-based materials, including our popular kits and libraries. This DNA-based materials...trying.  When we started in 2004, we were the plasmid repository. In 2016, we added viral vectors, and...addgene.org! Fun fact! Did you know you can search for plasmids from a publication by pasting the paper's title...Our first request was on September 8, 2004 (for pcDNA-p160MBP, if you're curious.) Since then, we've distributed...

...Episomal vectors- extrachromosomally maintained DNA plasmids that express reprogramming factors To date,...viral origin of DNA replication, OriP and the EBNA-1 sequence that encodes for the DNA binding protein...and to model diseases in vitro. Find stem cell plasmids at Addgene Many sources of adult somatic cells... amplification of the episomes concurrently with DNA amplification in the host cell. This enables relatively...group is an optimized mixture of three episomal plasmids (Table 1) encoding the reprogramming factors. ...enhance the reprogramming efficiency, an extra plasmid, pCXWB-EBNA-1 is used in the reprogramming cocktail...increases protein expression from the other episomal plasmids. When used on PBMCs, the pCXLE episomal vector...

...inhibiting crRNA loading, inhibiting DNA binding, or inhibiting DNA cleavage.   Five ways to anti-CRISPR...Bondy-Denomy J, Corn JE, Doudna JA (2017) Disabling Cas9 by an anti-CRISPR DNA mimic. Sci Adv 3:e1701620...proteins can also be used to prevent dCas9 tethering to DNA. Since these early anti-CRISPR studies, scientists...block CRISPR activity one of three ways: Inhibit DNA binding Example:  AcrIIA4 blocks Cas9’s interaction...crRNA-Cas complex (Zhu et al., 2019). Inhibiting DNA cleavage Example: AcrIIC1 binds to the HNH endonuclease...endonuclease domain of Cas9 and prevents target DNA cleavage (Harrington et al., 2017). Figure 1: Anti-CRISPR...CRISPR   Resources on Addgene.org Find CRISPR Plasmids for Your Research Interested in light-inducible...

...Read blog posts related to plasmid protocols Find protocols for purifying DNA without a kit Resources ...precipitate RNA, but not DNA and proteins. Lastly, using a DNase (there are several DNase enzyme products on...As with DNA isolation, scientists commonly rely on RNA isolation kits to make their life easier. Recently...Recently, we published a blog on DNA purification without a kit that outlined several reasons why doing ... one should take care whenever doing any type of DNA or RNA purification to avoid contamination, take ...RNA extraction. RNA is inherently not as stable as DNA- it is single stranded and its ribose groups are ...There are many alternative methods for isolating DNA without a kit. However, that isn’t the case for RNA...

...its method of DNA cleavage. Instead of cleaving DNA at a defined site to form a clean DNA break, Cas3 locates...sequence and initiates single stranded DNA degradation. Cas3 DNA digestion can proceed up to multiple kilobases...fidelity ssDNA targeting. RNA targeting Cas enzymes Move over Cas9, CRISPR isn’t just for DNA anymore!...highlighting their similarities and differences to Cas9. DNA targeting Cas enzymes Cas9 – The genome engineering...genome where it will proceed to generate a blunt DNA double strand break. Cas9 has been studied for genome...expanding number of others. Best for: a wide range of DNA edits.   Cas3 – The shredder What sets Cas3 apart...but it is effective at wiping out large regions of DNA and has anti-viral application potential as a result...

...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...genome-wide CRISPR inhibition Covalent tethering of DNA template to Cas9 SECURE base editors Nme2Cas9 CRISPR...CRISPR inhibition library Covalent tethering of DNA repair template to Cas9 Homology-directed repair ...Communications Biology Find the Cas9-PCV and PCV-Cas9 plasmids Selective curbing of unwanted RNA editing (SECURE...paper in mSphere Find the dCas9 and dCas9-Mxi1 plasmids If you have a new CRISPR tool you’ve recently...

...editing of a target base in genomic DNA without double-stranded DNA cleavage." Nature 533.7603 (2016): ...Programmable base editing of A• T to G• C in genomic DNA without DNA cleavage." Nature 551.7681 (2017): 464. PubMed...all CRISPR plasmids and resources at Addgene Find CRISPR base editors Find CRISPR plasmids for plants ...frequencies and relies on the efficient delivery of donor DNA to facilitate repair. Since the development of CRISPR-mediated...Cas9 nickase (nCas9). nCas9 creates a nick in the DNA by cutting only a single strand, dramatically reducing... detrimental indel formation. After binding with DNA, the CBE deaminates a target cytosine (C) into a ...this change. However, there are no naturally known 'DNA adenosine deaminases' which would theoretically give...

...other can the DNA strands take part in rolling circle DNA amplification. The amplified DNA segment is labeled...Fluorescent protein plasmids Use FRET to study protein-protein interactions Deposit plasmids with Addgene! ...Tagged protein can easily be overexpressed from a plasmid but overexpression may interfere with the system.... Both antibodies have a short antibody specific DNA strand added to them. Only if the proteins of interest...

...chromatin, the complex of DNA and proteins that forms chromosomes. These long range DNA interactions are often...digest cuts DNA into smaller pieces, and proximity ligation creates chimeric circles of DNA that are distant...CAPTURE method. After protein and DNA complexes are cross-linked, DNA is digested into smaller pieces. ...ligated together to create loops of DNA. This is key to linking pieces of DNA that are far away from each other...important supporting roles. Enhancers are cis regulatory DNA sequences that, when bound by transcription factors... looping with the photo “developed” by analyzing DNA sequencing data. While “pictures” generated with ...picture” is then developed by detecting these chimeric DNA fragments by PCR or next-generation sequencing. ...

...to defend against both DNA and RNA viruses - 2 cas systems regulate at the DNA level and 1 at the RNA ... Addgene’s experience helping scientists share plasmids, especially those of the CRISPR variety (see slides...is in one of the top 10 countries in number of plasmid requests from Addgene. Addgene - Canton Nucleic...Opening talks – Genomics discoveries and moving beyond DNA Eric Green, Director of the National Human Genome... inheritance that might be less error prone than DNA replication. He too mentioned how “surprising” small...ribosome is in a complicated conformation “not like DNA which has a boring structure.” That certainly summed... dna...

...fluorescent protein – you first need the DNA sequence that encodes it. That DNA sequence then needs to be incorporated...classrooms, like gel electrophoresis to separate DNA or transforming cells with green fluorescent protein...ll work with you to get the word out about your plasmids and their potential educational uses. We believe...30083609. PubMed Central PMCID: PMC6070313. Find the plasmids from this publication at Addgene! Additional ...developmental biology Use the pathways over time plasmids to engage students in functional genomics research...out our molecular biology reference pages Find plasmids from the BioBits BrightTM publication here ...

...thermocycling program. One key difference is the plasmid DNA must be released from the bacteria in order to...just need to lyse the bacteria to release the plasmid DNA by either briefly boiling the sample before use... sequences of the junctions between the plasmid and insert DNA. Colony PCR will greatly reduce the number...the correct orientation or even if it’s in your plasmid backbone. Backbone-specific primers: A second...orientation of your insert or if the insert is in your plasmid Have to make a new primer pair for each insert ...about the size of the insert and if it's in the plasmid Can be used to screen clones that were created ...created with the same backbone because primers are plasmid dependent Doesn't provide information about the...

...transfer plasmid, which contain regions of dsDNA. To generate a 3’ hydroxyl group needed for DNA replication... AAV.  Where do you start? Plasmids of course! You just need three plasmids to start making the AAVs you...packaging plasmid which contains the AAV structural and packaging genes, the adenoviral helper plasmid which...packaging plasmid and take a look at each of its parts. Key elements of the AAV packaging plasmid The packaging...packaging plasmid aka the RepCap plasmid encodes two genes: the replication (rep) gene and the capsid (... AAV RepCap plasmid! Check out this blog post to learn more about the AAV transfer plasmid! References... for the virus to replicate,  and the transfer plasmid which contains the viral genome. In today’s blog...

...) ChiRP-seq RNA Hi-C DNA (genome-to-genome interactions) DNase-seq DNA (regions that are accessible... regions of DNA that demonstrate “spooky action at a distance.” Through the marvel of DNA compaction, ...details on DNA compaction and the structural proteins that make it possible, see the appendix DNA is a 3D...See below for further details. DNA is a 3D Fractal Figure 2: DNA compaction. Graphics courtesy ...For instance (chr3, 1, 10) indicates a stretch of DNA at the very beginning of the third chromosome, ten...coordinates for genetic elements like protein, RNA, and DNA (Table 1). As with GPS in the real world, coordinates... examine how we can locate powerful stretches of DNA known as enhancers with the help of genome math. ...

...amazing plasmid technologies developed by our community of depositors. With over 40,000 plasmids avaliable...amazing new plasmid tools that have come through our doors in 2015. Do you have a favorite new plasmid tool ...system plasmids are available through Addgene and are already very popular. Use these plasmids to increase... can visualize: Cell Structure Plasmids: Cell Structure Plasmids: ER mCherry-Sec61 beta BFP-Sec61...luciferase reporter gene plasmid pGL3 pGL3 luciferase reporter gene plasmids have been used extensively...find all of our Hot Plasmid articles on our website. We update the our Hot Plasmids articles every quarter...Read Our CRISPR 101 Posts Read Our Plasmids 101 Posts Read All of Plasmid Technology Posts Resources at ...

...based on the functional elements they contain. Plasmid DNA in both prokaryotic and eukaryotic systems must...Download our Plasmids 101 eBook to get all the background you need to start working with plasmids! What are...Techniques Read all of our Plasmids 101 Posts Resources on Addgene.org Browse our Plasmid Reference Pages Just...Plasmids designed to express genes in a given host cell type are generally broken down into two broad...Rho-dependent terminators are not usually employed in plasmid-based expression systems, so these will not be ...the end.  Nearly all common bacterial expression plasmids use Rho-independent terminators, which include...the right. The tertiary structure of the hairpin-DNA complex is thought to destabilize the transcription...