...amount of spurious DNA from the packaging cell line, helper and packaging plasmids. We decided to use ... allows us to provide scientists with complete plasmid sequences. After launching our viral service in... the same frequency, we reasoned that, following DNA extraction, the [+] and [-] strands might naturally...restriction enzyme based methods, we showed that the DNA extract is a heterogeneous mix of single-stranded...double-stranded species. While not all of the molecules in the DNA extract associate with complementary strands, enough...identity of AAV genomes and detect contaminating AAV DNA. In addition, we wrote an open-source custom Python...Moullier P, Léger A (2015) Advanced Characterization of DNA Molecules in rAAV Vector Preparations by Single-stranded...

...sequence is a strict requirement for Cas9-mediated DNA cleavage. The need for more PAM sequences While...S. pyogenes Cas9 that were able to cleave target DNA sequences located upstream of either NGA or NGC PAM...increase the specificity of genome modifications at DNA targets adjacent to NGG PAM sequences when used in...VRER Cas9 variants are capable of cleaving genomic DNA in mammalian cells and zebrafish embryos, and they...284 . https://doi.org/10.1038/nature24049 Find plasmids from this paper at Addgene. Amrani N, Gao XD,... https://doi.org/10.1126/science.aaq0180 Find plasmids from this paper at Addgene. Kim D, Kim J, Hur...863–868 . https://doi.org/10.1038/nbt.3609 Find plasmids from this publication at Addgene. Kim E, Koo ...

...Agrobacterium contains a plasmid with a T-DNA (transfer-DNA) region. During infection this T-DNA region is transferred...transformation Read our Plasmids 101 blog posts Resources on Addgene.org Browse our Plant Plasmids and Resources...page Find synthetic biology plant plasmids Find CRISPR plant plasmids ... natural process by replacing the genes on the T-DNA region with our gene of interest or a reporter gene...brought into contact with the leaf tissue and the T-DNA region with the gene of interest is transferred into...localization of your protein of interest. Find all plasmids from the Lee-Parsons lab. Test different leaf...

... target both RNA and DNA, though it is largely believed that the cleavage of DNA is the primary function... foreign DNA into the CRISPR array. Lastly, type IV systems are most often found on plasmids. The many...Kyrpides, N. C., Banfield, J. F., & Doudna, J. A. (2018). Programmed DNA destruction by miniature CRISPR-...related immune mechanisms that could target foreign DNA with unique properties. This led to a need to develop...recently that researchers began building optimal plasmids to harness the power of Class 1 systems. Class...overall. Type I systems degrade large sections of DNA thanks to the dual action power of Cas3. Cas3 functions...helicase and a nuclease, unwinding and cutting up DNA after recruitment by Cascade. As you might imagine...

...on the Addgene Blog Plasmids for endogenous gene tagging in human cells Plasmids 101: Protein Tags Fluorescent... how proteins interact with one another (or with DNA) to perform diverse biochemical tasks in the cell...from many pitfalls: they require construction of plasmid vectors and/or rely on cumbersome in vitro synthesis...-IN” bioinformatics tool for guide RNA and donor DNA design, and implemented our optimised protocols into...to cells: a guide RNA, Cas9 protein, and a donor DNA that contains the tag sequence flanked by homology...CRISPR experiments (in collaboration with Integrated DNA Technologies, USA). These synthetic RNAs can also...csRNP complex formation, a single-stranded donor DNA (ssODN) that contains the tag-encoding sequence and...

...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles ...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...need. If you'd ever like to write about a recent plasmid deposit please sign up here.        New toolkit... the Kanemaki lab has created a new toolkit of plasmids for the N- or C- terminal tagging of proteins ... be from 2-6 base pairs in length and follow the DNA sequence targeted by the Cas9 nuclease. The Streptococcus...previously difficult. Find SpCas9 and Target-AID-NG plasmids at Addgene! Nishimasu H et al., Science, 2018....datasets for research and innovation. Find MerMAIDs plasmids at Addgene Opperman J et al., Nature Communications...

...chosen for both Cas9 and gRNA expression       Plasmid DNA may be incorporated into the genome     Off-...CRISPR plasmids) are not expressed. Because this method does not require the delivery of foreign DNA, and...commonly performed by transfecting cells with a plasmid, such as PX459, which encodes Cas9 and contains...targets with similar efficiency as compared to plasmid-based expression of Cas9/gRNA and can be used for...fluorophore tagged dCas9 may require the use of plasmid or viral-mediated delivery. You’ve decided to...and purify His-tagged Cas9 from E. coli using a plasmid such as pET-28b-Cas9-His from Alex Schier’s Lab...experimental systems that may not be amenable to plasmid based methods. One of the most common techniques...

...Nathaniel Roquet from the Lu Lab at MIT developed plasmid based systems that report the order in which specific...technology that can be used to quickly barcode a set of DNA sequences. In this post, Alan Wong (formerly of the...and the associated publication. Optimizing Donor DNA for Enhanced CRISPR Genome Editing CRISPR has revolutionized...CRISPR, it isn’t always easy to insert a section of DNA into the genome of your target organism. In this ..., describes how you can better design your donor DNA template to achieve more efficient genome editing...direct a variety of customizable functions to your DNA sequence of interest. Potential applications of the... plasmid...

...efficiency of plasmid delivery to a cell. In contrast, TREE provides a read out for plasmid delivery and...Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551:464–471 . https://doi.... to CRISPR/Cas9 edits that rely on double strand DNA breaks. Due to this inefficiency it is crucial for...enzymes. At the same time Cas9n cleaves the opposing DNA strain which results in two DSBs that can be repaired...panel of eGFP reporters to quantify the on-target DNA editing efficiency of APOBEC-Cas9 editisome complexes...circumvents the need for DSBs in monitoring on-target DNA editing efficiency of APOBEC-Cas9 editisome complexes...after being targeted by a cytidine deaminase base DNA editor. Specifically the BFP mutant (BFPH66) contains...

...number of plasmids found in each cell, but also whether the plasmid gets integrated into the host DNA or is...types of yeast plasmids are defined below: Yeast Integrating plasmids (YIp): These plasmids lack an ORI...our first few Plasmids 101 posts, we focused mainly on the elements required for plasmid maintenence within...integration. Yeast Episomal plasmids (YEp): These are most similar to bacterial plasmids and are considered “...well-defined genetic system, and a highly versatile DNA transformation system.Unlike most other microorganisms...simple gene replacement/mutation. Yeast expression plasmids used in the lab typically contain all the necessary...how the regulation of bacterial ORIs determines plasmid copy number within the bacterial cell. Similary...

...your expression plasmid from these strains is not recommended as these auxillary plasmids may be isolated...control such as the pLys plasmid, which suppresses basal T7 expression. The pLys plasmid contains a chloramphenicol...arabinose-inducible plasmid will not express in an IPTG induction strain for example, nor will a p15 plasmid be compatible... for More Plasmids 101 Articles?  Find them all here: http://blog.addgene.org/topic/plasmids-101   ...In a previous Plasmids 101 blog, we reviewed the salient features of several popular strains of E. coli...coli for DNA propagation. While great for cloning purposes, these E. coli strains are not usually well...strain? Protein expression from high-copy number plasmids and powerful promoters will greatly exceed that...

...achieved at the level of the DNA construct, where your RNAi-target is fused to the DNA backbone that contains...for mammalian RNAi Browse all plasmids for RNAi Check out Addgene's plasmids and resources for plant biology...biological function in a direct or unbiased way at the DNA, RNA, or protein level. The “correct” choice of tool...Therefore, techniques that function downstream of DNA, such as RNA interference, can reversibly exert their...predominantly used to disrupt function at the level of DNA, thereby limiting CRISPR’s effectiveness at high ...initial considerations, such as the starting material (DNA-based vector or purified RNA) and the initial state...typically hairpin RNA, or artificial microRNA). Using a DNA template for the RNAi trigger is the most prevalent...

...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 ...Alicyclobacillus acidoterrestris (AacCas12b) exhibits optimal DNA cleavage at 48 °C, a temperature incompatible to ...at 37 °C but preferentially nicks the non-target DNA strand. The lab then found gain-of-function mutations...paper in Nature Communications Find the Cas12b plasmids  Plant base editing with a Cas9-adenosine deaminase...wheat. Read the paper in Genome Biology Find plasmids containing these base editors Spatially and ...Neuroscience Find the AAV-based SaCas9 genome editing plasmids String assembly gRNA cloning (STAgR) String ...

...regions containing the putative tracrRNA into a plasmid and expressed it in an E. coli bacteria strain....References 1. Harrington, Lucas B., et al. "Programmed DNA destruction by miniature CRISPR-Cas14 enzymes." Science...sheet Resources on Addgene.org Find the Cas14 plasmids here Checkout our CRISPR guide page Browse the...indiscriminately cleave ssDNA in trans like Cas13 and Cas12 do for RNA and dsDNA, respectively. However...activated. Cas14 in diagnostics By cutting ssDNA but not dsDNA Cas14 is not a good candidate for a new genome-editing...combination enabling the detection of RNA, dsDNA, and now ssDNA. By comparing the ability of Cas12 and Cas14...and maybe more efficient Cas systems, Jennifer Doudna’s lab analyzed metagenomic datasets to try to determine...

... in E. coli with two plasmids (Figure 3 below): Input Plasmid: The input plasmid (high copy, ColE1 origin...inducible promoter (PPhlF).  Output Plasmid: The output plasmid (single copy, bacterial artificial chromosome...through Addgene. Their plasmid maps and GenBank files can be found on their plasmid pages. All two-input...indicated figures in our paper (1)) Input Plasmid Output Plasmid Fig. 3A pNR64 pNR160 Fig. 4A pNR220...state machines (RSMs), which take on a particular DNA-sequence configuration (state) based on the identity...defined by the sequence within a prescribed region of DNA termed the “register”. Each chemical input to an ...result of their recombination is the excision of the DNA between the sites (Fig. 1B below). Alternatively,...

... the Doudna lab has used Cas12a’s non-specific ssDNA degradation to develop the method termed DNA Endonuclease...specific DNA sequence, such as the Human papilloma virus (HPV) genome, via a crRNA. An ssDNA-fluorescently...signal when the ssDNA is degraded, is added to the reaction. To enhance sensitivity, the DNA is first amplified...quantified to determine the presence and quantity of DNA, RNA or a mutation of interest. Together SHERLOCK... increase the sensitivity of the assay, targeted DNA or RNA from a sample is first amplified using RPA...coupled with T7 transcription to convert amplified DNA to RNA for subsequent detection by Cas13. This amplification...with the ssRNA reporter enables SHERLOCK to detect DNA or RNA with atomolar sensitivity and single base ...

...rescue! Base editors chemically modify DNA bases without breaking the DNA backbone, sidestepping the problem...uracil without cutting DNA. Uracil is then subsequently converted to thymine through DNA replication or repair...unmodified DNA strand so that it appears “newly synthesized” to the cell. Thus, the cell repairs the DNA using...strand of DNA and converts a C to U without producing a double strand break. UGI = uracil DNA glycosylase...Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature, 551(7681), 464–471. https...editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 533(7603), 420–424...homology-directed repair (HDR), the pathway for resolving DNA double-strand breaks (DSBs) preferred by researchers...

...incorporated into the DNA during DNA synthesis and then diluted with subsequent rounds of DNA synthesis (cell... for panel design. Find streptavidin plasmids and biotin plasmids at Addgene!     Figure 1: Indirect...found within the cytosol/nucleus, or visualizing DNA instead of proteins. Some stains can diffuse through...targets include transcription factors, histones, and DNA repair enzymes. Note that when using the nuclear ...Dye stains are non-antibody-based stains that bind DNA or free amines of proteins. Depending on the application...previously induced gene knockout. Besides amine- and DNA-binding dyes, nucleoside analogs like bromodeoxyuridine...stains are membrane-permeant or -impermeant and bind DNA or free amines, depending on the application. Paul...

...PubMed PMID: 26950006 Plasmid ID Plasmid Name Plasmid Type 74309 pcDNA3.0-TRPV4-p2A-ferritin-p2A-mCherry...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief articles...features and applications of a partiular plasmid or set of plasmids. We hope that these articles make it ... you to find and use the plasmids you need. You can find all the hot plasmids from 2016 below. With over...over 45,000 plasmids, we can't write posts for every great plasmid that comes into the repository, but...write about your plasmids in a future blog post. No time to read? Listen to our hot plasmids segment on the...alone involves much literature searching, DNA synthesis, and plasmid assembly. Luckily, researchers from Douglas...

... ITRs are the elements that make an AAV plasmid an AAV plasmid.  Within the AAV genome, ITRs serve as...they are in the same DNA molecule as the gene that they package. In a transfer plasmid, the transgene is ...the single stranded DNA genome of AAV in the native virus. In an AAV vector plasmid, they flank the gene...growth culture and result in a high percentage of plasmid DNA with mutated ITR sequences. Using recombinant-deficient...gene of interest in a cell or animal model. AAV plasmids contain several important elements, but among ...located immediately outside of the ITRs in the plasmid backbone and do not properly assemble, but these...