...the opposite strand). ABEs make an A to G change (or a T to C change on the opposite strand). Both are ...place on the free strand of DNA and converts a C to U without producing a double strand break. UGI = uracil...repair (HDR), the pathway for resolving DNA double-strand breaks (DSBs) preferred by researchers. Compared...are base transition editors: converting one purine base to the other or one pyrimidine base to the other...other. Figure 1: Base transition editors catalyze purine-to-purine or pyrimidine-to-pyrimidine base... indicate the change introduced to the opposite strand following base editing and DNA repair. Black arrows...BioRender.com. How does base editing work? Base transition editing requires three elements. They are, broadly...

...Biological Material Transfer Agreement (UBMTA) is one of the most commonly used ones for transfers between academic...for more information. Happy transferring!    Many thanks to our Tech Transfer team members Maura Coughlin...Addgene, then you’ve been involved in the material transfer agreement (MTA) process. Even though this process...curious about the hows and whys of the material transfer agreement, read on!  What is the MTA?  An MTA...the creation of a standardized agreement on the transfer of biological materials in 1995: the UBMTA. Because...most academic and nonprofit institutes, the Tech Transfer department is responsible for managing research...plasmid with Addgene, your organization’s tech transfer office is sent a deposit agreement. The One-Time...

...also incorporated LOV-Turbo into TransitID experiments.  Find TransitID plasmids here! These two developments...genetic cargo Optogenetic proximity labeling and TransitID Tracking cellular history with protein ticker ...in vitro and in vivo. Incorporating reverse transcriptase (RT) variants, PE6a and PE6b are 500–800 bp ...be tethered on the viral surface, expanding the range of targetable cell types. DIRECTED is compatible...37612276.   Optogenetic proximity labeling and TransitID By Mike Lacy Proximity labeling can identify protein...plasmids here! Next, Qin and Cheah et al. described TransitID, where two proximity labeling enzymes, TurboID...catalyzing the biotinylation of nearby proteins. B) TransitID uses sequential labeling by TurboID and APEX2 ...

...of Pennsylvania Vector Core. Transfections are performed using the transfer plasmid, a plasmid encoding...PCR with primers targeting the transfer plasmid used in the transfection. PCR products are analyzed by ...cell cultures are inspected for sterility. Transducibility Some viral vectors are tested in vitro and ...images).     Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre (55634...filtration through a 0.45 µm polyethersulfone (PES) membrane. The preparations are then aliquoted, frozen, ...from Wang et al., 2018). Briefly, 293T cells are transduced with serial dilutions of a lentiviral vector,...taken offline and discarded. Confirmation of the transfer plasmid Addgene uses a rigorous barcode matching...

...fused to the N-terminus of LSSmOrange pLSSmOrange-C1 Orange Mammalian Express a gene of interest fused...GFP variant) commonly used with mRuby2 pLSSmOrange-N1 Orange Mammalian Express a gene of interest fused...Bioluminescence Resonance Energy Transfer CRET= Chemiluminescence Resonance Energy Transfer    Interesting Articles...FRET, aka Fluorescence (Förster) Resonance Energy Transfer, technique which allows the detection of molecules...distance-dependent physical process by which energy is transferred non-radiatively from an excited molecular fluorophore... (the acceptor) by means of intermolecular long-range dipole-dipole coupling." The emission of the acceptor...fused to the C-terminus of LSSmOrange mRuby2 Red Mammalian Expresses mRuby2 (a RFP variant) commonly used...

... technologies have transformed the palette of experiments possible in a wide range of organisms and cell...genomic locus (CRISPR-ChAP-MS) could further transform the range of experiments imaginable in C. elegans. ...Genome Editing with Short Single-Stranded and Long, Partially Single-Stranded DNA Donors in Caenorhabditis...at 25 ºC. This method resulted in efficiencies ranging from 11-100% of F1 animals carrying precise knock-ins...This stringent selection allowed me to optimize a range of editing parameters: oligo repair templates with...with homology arms of 35-80 bp, and DNA double-strand breaks up to 54 bp from the desired insertion site..., they devised guidelines for designing single-stranded repair templates (Farboud et al., 2019).   The...

...flow of electric charges across cell membranes and maintain membrane potential in response to visible light...across the axon's membrane: once a certain number of positive ions crosses the cell membrane, a threshold ...rhodopsins enable ions to flow across the axon’s membrane, thereby controlling neural activity (Bernstein...the opsin into the correct orientation to be transcribed (Figure 2) (Sharma and Zhu, 2014). How does FLEx... optogenetic FLEx vectors in combination with transgenic animals or rAAVs expressing Cre under a specific...the mechanisms of sleep and anesthesia. In Cre transgenic mice, they targeted the dopamine (DA) neurons... vectors combined with the use of several Cre transgenic mice targeting different brain areas enabled ...

... pair has focused considerable attention on transcription factors, starting with an established method... relies on antibodies to recognize and bind transcription factors directly, but that’s a slow-going process...ENCODE project is to look at all 1,500 or 1,600 transcription factors and figure out where they bind to the... ChIP-seq, but it’s really hard for lots of transcription factors. Our groups got together to adapt CRISPR...big difference. We can now look at many more transcription factors. Addgene: How does the system work? ...second plasmid to target an epitope tag onto the transcription factors. By the way, you can actually use this... in all kinds of different assays, not just transcription factors. The second vector is a homology directed...

...A double-strand DNA break occurs within the core spacer of each loxP site and the two strands are ligated...maintained).  Translocation: If the sites are on separate DNA molecules, a translocation event is generated... expression – placing a series of transcriptional and translational termination features with loxP sites... pun) specific, targeted DNA modifications in transgenic animals, embryonic stem cells, and/or tissue-...that there is virtually no chance of it occurring randomly. Therefore, inserting loxP sites at deliberate...Inversion: If the loxP sites are on the same DNA strand and are in opposite orientations, recombination...research tool, especially in the field of mouse transgenics. A few of the most common uses are listed below...

...et al., 2018). Unbiased, random insertions can be generated with a transposon-based strategy (Sheridan...fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction." BMC neuroscience... This type of interference is known as steric hindrance. For instance, GFP may occlude catalytic sites...sites or motifs that are necessary for post-translational modifications. Despite these potential issues...targeted to the endoplasmid reticulum based on the appearance of OSER whorls. This assay is quantitative and...various fluorescent proteins (Botman et al, 2018; Cranfill et al, 2016; Lambert, 2019). These characteristics...applications, many were concerned with steric hindrance by the fluorescent protein. Thus, scientist began...

...which ensures the editor will only be transiently present in transduced cells, limiting off-target editing... dCas9 fused to a DNA methyltransferase — that can drive lasting transcriptional silencing (Nuñez et al...virus-like particles (eVLPs) offers an option to transduce cells directly with ribonucleoprotein cargo. eVLPs...attB/P recombinase landing sites on opposite DNA strands. When the prime editor releases the DNA, the two.... (2024). Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes...Histone tail for Autoinhibition Release of Methyltransferase (CHARM), with several advantages over the ...  Rather than overexpressing an exogenous methyltransferase for epigenetic silencing, which can be toxic...

...variants (hereafter referred to as Cas9) and a single stranded guide RNA (gRNA) is sufficient to modify the genome...variants have been created in an effort to increase translation in a particular plant species or cell type (Belhaj...CRISPR components can be expressed stably or transiently depending on the delivery method and cell type...CRISPR components can be delivered and expressed transiently using a standard detergent, Polyethylene Glycol... More information on Agrobacterium-mediated transformation can be found in this blog post. The pDGE Dicot...Simplified schematic of agrobacterium-mediated transformation in plant cells. Agrobacterium tumefaciens can...plant genome. B) The Ti plasmid and helper are transformed into agrobacterium tumefaciens and exposed to...

...into smaller pieces, run on a gel, and transferred to a membrane for analysis through hybridization of ...researchers generating transgenic animals may choose Southern blotting to screen founder transgenic animals for ...techniques used to generate transgenic mouse lines, such as BAC transgenic or traditional knockins/knockouts... control sample. Southern blotting applications range from confirming the results of cloning or knockout...for the inserted transgene because Southern blots have a lower false positive rate than real-time PCR (4...hydroxyl group required for the extension of a DNA strand (8). Adding a small amount of a single radiolabelled...regular nucleotides results in the synthesis of DNA strands of all possible lengths because extension is terminated...

...proteins, which are directly trafficked to cell membranes. Membrane trafficking of farnesylated derivatives enables...Brainbow-3.0, but with mOrange2, EGFP and mKate2 as the fluorophores; mOrange2 is the default state. In... virus post-transcriptional regulatory element (WPRE), commonly used to increase transgene protein levels...fluorescent proteins: RFP (red), YFP (yellow) and M-CFP (membrane tethered cyan). Without Cre recombination, RFP...first fluorescent protein in the construct, OFP (orange), is expressed when Cre is absent; stochastic Cre...This system differs from Brainbow-1.0 in that transient Cre recombination is required to prevent constant... both fluorescence and sequence overlap (coral mOrange2, jellyfish EGFP, and sea anemone mKate2.) They...

...this FRET you speak of? Förster Resonance Energy Transfer (FRET) was originally described by Theodor Förster...alternative acronym Fluorescence Resonance Energy Transfer. Unlike the typical excitation and emission of...excited fluorophore, FRET involves a non-radiative transfer of energy (i.e. no emitted photons) from the excited...excitation by absorption of a photon 2. Energy transfer from the excited donor directly to the acceptor...the acceptor fluorophore. Otherwise the energy transferred from the donor will not be able to excite the...other and be oriented appropriately for energy transfer via dipole-dipole interaction. The efficiency ...biological processes occur within the typical FRET range, FRET efficiency is used to infer an interaction...

...process known as bioluminescent resonance energy transfer (BRET). The fluor then emits light at a tissue-penetrating... consists of NLuc fused to the more penetrative orange fluorescent protein fusion (OgNLuc). Both fluorescent...for excitation. The result was the cyan-excitable orange fluorescent protein, CyOFP1. CyOFP1 was molecularly...and improved maturation time compared to similar orange fluorescent proteins. In their work, the authors...great imaging tools generate cyan, yellow, green, orange and red fluorescence. The paper reports a wide ...Plasmid #85199), Yellow (YeNL; Plasmid #85201), Orange (OeNL; plasmid #85202) and Red (ReNL; plasmid #85203...have been made using fluorescent resonance energy transfer (FRET) probes. These probes generally consist ...

...concentration lies within the dynamic range of the curve. The dynamic range is the linear span between the lowest...the dynamic range of your standard curve, so if your sample does not fall within this range, it may be ... of the series dilutes by 1:2 for a series that ranges from 1:2–1:32. The specific dilution series used...Figure 1: A 1 mg/mL control is diluted in a series ranging from 1:2 to 1:32. Each individual step of the series... how well the data points fit the trendline. R2 ranges from 0 to 1 with 1 being a perfect fit and 0 indicating...that the curve can accurately measure.  Dynamic range depends on a number of factors, including the dilution...being used to measure the response. The dynamic range needs to be wide enough to cover all possible concentrations...

...members to the Board of Directors: Khandan Baradaran, Frank Stegmeier, and Luk Vandenberghe. We’re excited...members and get to know them! Khandan Baradaran, PhD Khandan Baradaran’s expertise in viral safety, gene therapy... with health authority regulations,” Baradaran says.  Baradaran’s path to science began in high school... getting these reagents is profound.” Frank Stegmeier, PhD Frank Stegmeier is the Chief Scientific Officer...in viral genetics. In regards to open science, Baradaran thinks the best way to solve problems is to work...think will get the research funded,” she says. Baradaran met Addgene Executive Director Joanne Kamens several...the targets that have the biggest potential to transform patients' lives. Previously Stegmeier was the ...

...72 hours post-transformation/transfection in a transient assay. For stable transformation, regenerated ...Agrobacterium-mediated transformation. We also constructed smaller vectors suitable for transient assays using ...changes a single base with an efficiency typically ranging from 25-75%, while the success of precise change...creates a nick in the DNA by cutting only a single strand, dramatically reducing the likelihood of detrimental...of the desired T-A base pair. Figure 1: Transition mutation (C>T, G>A, A>G, and T>C) installation...deaminases (ecTadA), fused it with dCas9, and transformed it into E. coli harboring the defective CamR ...using protoplast transformation. Cytosine base editors for plant There are several CBEs available for plant...

... affecting protein translation of different reporter proteins. Protein translation requires the start ...compared to CRISPR/Cas9 edits that rely on double strand DNA breaks. Due to this inefficiency it is crucial...constitutively expressed to identify successfully transfected cells. C-to-T editing by the APOBEC-Cas9 editosome...that results in the restoration of fluorescence. Transient reporter for editing enrichment (TREE) The Wang...Wang and Brafman lab developed a transient reporter for editing enrichment (TREE) to purify single base... to cells segregated solely by a reporter of transfection, which only report the efficiency of plasmid...frequency of base edits of interest than solely transfected cells. “Gene On” (GO)- a functional reporter ...

...attractive due to the ease of transduction in difficult-to-transfect cell types ranging from primary to embryonic... traditional transfection and random genomic integration of a plasmid. Viral transduction is also generally...integrated into the genome of the target cell or transiently expressed. It’s important to note that for all...vector can express your construct in target cells transiently or constitutively depending on the virus used...generally less toxic to cells than transfections, preserving cell viability. Library delivery Screening... entire genome, overexpression of all human transcription factors, barcoded libraries to trace cell lineage... with viral vectors instead of a traditional transfection method. This is because viral systems have a...

...Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences...easily manipulated with tools like CRISPR to make transgenic lines. The fruit fly (Drosophila melanogaster... in only 6 weeks compared to months to make a transgenic mouse line. Over the years the fly has become...DNA or RNA to edit their genomes or to create transgenic animals. Zebrafish are an ideal model organisms...development studies as their embryos are completely transparent. Thus scientists can easily observe early developmental...can be difficult in other vertebrate species. Transparency also allows scientists to easily observe fluorescently... control the temporal expression of different transgenes, the Stainer group determined that there are ...

...The development of the transient transfection platform for assaying transcriptional activity, for example...such as p300 and CBP histone acetyltransferases or CARM1 methyltransferase. This attendant signaling feature...dietary origin) that are diffused or actively transported from cells, traverse the body through the bloodstream...ligands NRs represent the largest family of transcription factors found in metazoans. The superfamily ... govern expression of genes involved in a broad range of reproductive, developmental, metabolic, and immune...multiple cell signaling systems. Activation of membrane receptors and signaling cascades conceivably allows...profiling datasets in to our data mining tool, Transcriptomine. We are currently nearing the completion of...

..., expressing Ad E1A and E1B, can be transfected with your transfer plasmid, carrying your gene of interest... performed 12–18 hours post-transfection to help remove toxic transfection reagents and keep the cells...helper plasmids, commonly called “triple plasmid transfection” (Figure 1).    Figure 1: Overview of key steps...cells happy. Harvesting virus Once you have transfected your cells, wait 2–5 days and then collect the supernatant...the concentration of viral particles that can transduce cells, which can vary based on target cells and... capsids, thus leading to an overestimate of transducible units in the virus preparation.  rAAVs  Now...Whitney, O., Walther, N., & Maurer, A. C. (2023). Transgene Expression in Cultured Cells Using Unpurified ...

...respondents (transfection method, amount of each component per transfection etc.). Take Pooran's NgAgo Survey...Doudna Lab Many thanks to our Guest Blogger Pooran Dewari! Pooran Dewari is a postdoc at the MRC Centre for...TOOL This post was contributed by guest blogger Pooran Dewari. Any views in this post are those of the...an ssDNA guide. Therefore, researchers must co-transfect cells with 5’-P-ssDNA guides and an NgAgo expression...delivery methods. Researchers can conveniently transfect/inject cells with in vitro preassembled Cas9/gRNA...). View the Complete and Up-to-Date Results of Pooran's NgAgo Survey The take-home message While most ...researchers will have to carry out repeated transfections with the ssDNA guide, although many may find...

...target and nontarget DNA strands upstream of the PAM site found in the nontarget strand. Protein Cas: ...sequences and keeping the array organized. Target strand: The strand of foreign DNA that Cas proteins recognize...recognize using an RNA guide. Non-target strand: The paired strand of foreign DNA cut by Cas9, whose sequence... and variable spacer sequences. The array is transcribed and processed into crRNAs, each including one...protospacer-adjacent motif” present in the non-target strand of DNA to be cut, which varies between CRISPR systems...from Addgene!   RNA crRNA: “CRISPR RNAs” are transcribed from the CRISPR array and include a spacer and...track down its suspect—the target DNA. tracrRNA: A “trans-activating CRISPR RNA” base pairs to the repeat ...

...between the edited strand and wild-type strand of the host DNA. When these strands hybridize, the nuclease...nuclease can detect strands with mismatches and cleave them. The results are then visualized using gel electrophoresis...is also key to perform these validations as translational researchers begin to use CRISPR as a therapy... search criteria are not exhaustive. Mismatch tolerance settings are often limited to off-target sites...nucleotides) that may still lead to off-target double-stranded breaks (Tsai et al. 2015). Additionally, current... genome-wide, unbiased identification of double-strand breaks (DSBs) evaluated by sequencing (GUIDE-seq...et al. 2013). GUIDE-seq and BLESS detect double-stranded breaks and do not require high sequencing read...

...different DNA strand comes along, it can attack this covalent bond thus joining the two DNA strands and releasing...planning to transform right away OR you can store the reaction at -20C overnight. 4. Transform TOPO Cloning...DNA sequence 5´-(C/T)CCTT-3' and digests double stranded DNA at this sequence. The energy from this breakage...stored as a covalent bond between the cleaved 3′ DNA strand and a tyrosyl residue of topoisomerase I (1). If...researcher to perform blue-white screening after transformation - self joining of the vector ends results in... minutes will not significantly improve the transformation efficiency).  5. Select and Analyze 10 White...add will depend on whether you are planning on transforming your reaction into chemically or electro-competent...

...hiPSCs for clinical purposes: mRNA transfection- this allows transient expression of the reprogramming factors...cells, thus expressing additional EBNA-1 transiently. The transiently expressed EBNA-1 increases protein expression...insertional mutagenesis, which is the integration of transgenes into the genome that can cause disruption of ...express reprogramming factors To date, the mRNA transfection method has not been used successfully to derive...using PBMCs as the source of somatic cells. This translates to only about 16 hiPSC colonies per 100 ml of...such low efficiency of reprogramming is a major hindrance in using PBMCs as a source of somatic cells for...system thus provides a practical way to generate transgene-free, virus-free clinical grade hiPSCs from easily...

...lentiviral vector to be transported retrograde and also has higher neuronal transduction efficiency and a better...virus to either restrict or broaden the host cell range (tropism). Pseudotyping is only done on enveloped...Lentiviral production uses three plasmids: (1) The transfer plasmid, (2) the envelope plasmid, and (3) the...the packaging plasmid. These are transfected into target cells which produce lentiviral particles.   ... titer is one of the reasons VSV-G is used to transduce stem cells and neurons. However, VSV-G’s broad...for off-target infectivity as it can bind and transduce many cell types before reaching the target cells...find tables and information on different viral membranes, tropism, and advantages and disadvantages: Table...

...scientists to quickly and easily generate zebrafish transgenic lines to study neurobiology and neuorodgenerative...pluripotent stem cells) through the expression of transcription factors OCT3/4, SOX2, KLF4, and c-MYC is the... method for generating iPSCs to express the transcription factors from a polycistronic cassette. However...comparable experimental results. Using PiggyBac transposons, the Woltjen Lab compared different variants ...reprogramming discrepancies. The polycistronic piggyBac transposon vectors tested in this work are available at ...approach Genetically modified organisms like transgenic mice are important tools for understanding the...a donor in FRET (fluorescence resonance energy transfer) and FLIM (Fluorescence Lifetime Imaging Microscopy...

...diderms, have a thinner peptidoglycan membrane and an outer lipid membrane. Common gram negative bacteria include...within their membrane, while the stain is washed from the gram negative bacteria’s thinner membrane. Procedure...destroys the outer lipid membrane, and the thin inner peptidoglycan membrane is unable to retain the stain...expanded on Gram’s discovery, adding a second stain, safranin, as a counterstain to help visualize the bleached...or monoderms, have a single thick peptidoglycan membrane. Common gram positive bacteria include staphylococci...and immediately rinse. Flood the smear with the safranin counterstain for 30 - 60 seconds. Rinse the slide...interaction of CV+ and I- in the thick multilayered membrane of the gram positive bacteria. In a gram negative...

... of the four strands of the cleaved duplex, while the fourth strand (the released strand in Figure 1 below...discovered that single stranded donor DNA: Complementary to the released strand, 127bp in length, With...single or double stranded donor DNA with other design parameters. Example single stranded donor DNA and ...presented in Figure 2. Figure 2: Single-stranded donor DNA interaction with the Cas9-DNA duplex...accessible to anneal with complementary single stranded DNA. We thus wondered if such a structure could... that are an order of magnitude greater than transfection approaches. Moreover, the cost of reagents is...relatively modest, requiring expressed Cas9 protein, transcribed sgRNA, and optional donor DNA. Chris Richardson...

...AAV5, AAV8) enable different patterns of transduction and transgene expression across different cell types..., Rumpel S. Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5...expressed from your cell line of choice after transduction with this virus. While various Cas9 mutants ...classic Cas9 nuclease, which introduces double stranded DNA breaks, can be expressed from lentiCas9-Blast...single stable Cas9-expressing cell line and then transduce this line with lentiviruses expressing various...experiments and over time (as compared to separate transductions with a single-vector system). Tips for using...is used to attain cell-specific expression in transgenic animals, where cre expression is restricted to...

... Retroviruses are single-stranded RNA viruses that use reverse transcriptase to synthesize complementary...for most experimental needs, and can transduce (infect) a wide range of cell types with a high efficiency... such as a heterologous envelope or a post-transcriptional regulatory element can be used for either type...Adeno-associated virus (AAV) is a small, single-stranded DNA parvovirus. AAV can infect humans and some...response as retroviruses. This virus can also transduce dividing and non-dividing cells, making it conducive...issue. Adenovirus Adenovirus has a linear, double stranded DNA genome which is non-enveloped. It can infect...Adenovirus is an effective option if you need high, transient expression of a genetic cargo or have a very large...

... that determine membrane expression and plasma membrane localization of transmembrane proteins, like rhodopsins...capable of broad transduction of the nervous system which eliminates the need for intracranial injections. ... to perform in vivo optogenetic experiments. Intracranial injections of AAV are commonly used to deliver...channelrhodopsin-based optogenetics minimally-invasive, Frances Arnold’s lab and Viviana Gradinaru’s lab used machine...neurons and mouse brain slices collected following intracranial injection of an AAV expressing a ChRger. In ...performed well in a tissue culture dish, that doesn’t guarantee their success in vivo. Therefore, the Arnold and...their cage. Since dopamine is an important neurotransmitter that promotes reward-motivated behavior, if...

...SLC6A8 mutations in Creatine Transporter Deficiency Creatine Transporter Deficiency (CTD) is a rare X-... to be transported from the blood and cross the blood brain barrier using creatine transporters. However...However, mutations in the transporter SLC6A8 impede this transport and result in global creatine deficiency...Accelerate Research Discoveries) Challenge provides grants for rare disease research. This year is our fourth... the SLC6A8 gene, also known as the creatine transporter. The Association for Creatine Deficiencies (ACD...GPCRs) detect molecules outside of the cell and translate those signals into cellular responses. For example...consist of many parts that work together on the cell membrane. Germline mutations in one of these parts, the...

...bacterial transformation One protocol that has been automated is high throughput transformation. With our... to do up to 96 transformations in one run compared to doing about 50 transformations in the same amount...easier and more efficient. Here, I’ll discuss transformation, DNA purification, and filling vials with agar...deposits (over 100 deposits per month!), manual transformations were taking a lot of time. Enter the Biomek... We wanted a machine that would improve our transformation protocol and, after looking at a few options... - one pod can pick up 96 tips for easy sample transfer between 96 well plates. The second pod can pick...out your samples. This can be useful if you’re transferring samples from 96 wells plates onto agar q-trays...

... copy. The coding sequence for GFP is transcribed and translated along with the gene, producing both a...exon of the gene to ensure it would be transcribed and translated. For an N-terminally tagged protein, ...destroyed by CRISPR/Cas9 during transfection. Making the double strand break and introducing the tag through...base pairs on each side, in fact. Later, when we transfect the cells, these large regions of homology enable...directed repair (HDR) process following the double strand break CRISPR/Cas9 makes at the target site. The...technique that briefly destabilizes the cell’s outer membrane and allows the components to physically enter ...go to work. Figure 3: Inducing the double strand break and introducing the tag. Created with BioRender.com...

...selectable, it can be transferred to a different genetic background via P1 transduction. Plasmid Expressing...products: 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the dsDNA was short enough...enough, a ssDNA whose entire complementary strand was degraded. Beta: Beta protects the ssDNA created by...higher recombination frequency when the lagging strand of DNA is targeted, it’s best to determine the ...design an oligo that is complementary to the lagging strand. In practice, it’s easier to not determine the ...replication and instead design oligos that target both strands. One of the two oligos will recombine with a higher...you’ll find out empirically which is the lagging strand. ssDNA substrate is more efficient than dsDNA with...