...gRNA in one AAV and a repair template in second a second AAV to deliver their repair template to mouse zygotes...two halves of a repair template spread across two AAVs, it is possible sequentially repair a target site...deliver a repair template that would take up ~6.5 kB if delivered in a single AAV. They report roughly ...Russell and Hirata from the University of Washington reported a surprising finding - when delivered via AAV ... mutations through aberrant double strand break repair, it has proven more challenging to develop high... via homologous recombination with a homologous repair template. It is this application - the creation...appropriate for a CRISPR-based approach. Second, the AAV preps generally used for gene editing are low titre, and...

... derived from Streptococcus pasteurianus  or the type II CRISPR3 module of Streptococcus thermophilus ...tools for your research! This time: Optogenetic repression in yeast Base editing CRISPRi in Caulobacter ... CRISPR-based DNA demethylation Optogenetic repressors of gene expression in yeast Megan McClean’s lab...generated an optogenetic toolkit that controls repression of gene expression in Saccharomyces cerevisiae...consists of dCas9, the mammalian transcriptional repressor Mxi1, and an optogenetic tool that controls nuclear...complex enters the nucleus where dCas9-Mxi1 can repress gene expression. In the dark, the NLS is inaccessible...Molecular Bioengineering Find the optogenetic repressor plasmids Cytosine base editors with minimized...

...other services that we provide, including viral preps and pooled libraries. As anyone who has ever worked... libraries. From start to finish the library preparation process only takes about one day, even for six...plasmids)! These libraries are then QC’d, pooled, and prepared for Illumina sequencing on our newly donated MiSeq... are “failed” and removed from the plate to be prepped again. These holes are then filled with other samples...VGS). QC is performed throughout the library preparation process and includes Picogreen quantification...often find a few mismatches in the origin of replication or other common backbone elements. Since we’ve...ve successfully grown the plasmid in culture to prepare it for sequencing, we feel confident that these...

...available as plasmids in the repository, as part of our in-stock viral prep service and can be selected...al., 2023). AAV1 capsids are available in the repository and can be selected as a serotype for Addgene...AAV1 tools are available through our in-stock viral prep service.  AAV2 Despite being isolated second, AAV2...AAV2 capsids are available as plasmids in the repository and can be selected as a serotype for Addgene...AAV2 tools are available through our in-stock viral prep service.  AAV5 AAV5 is the most genetically distinct... targeting astroglia. It has additionally been reported to have tropism for lung, liver, vascular endothelial...al., 2023). AAV5 capsids are available in the repository and can be selected as a serotype for Addgene...

...involves viral vectors and plasmids with repeating sequences. Repeating sequences within a plasmid can undergo... (AmpR) and origins of replication (ori and f1 ori) which allow it to replicate.   How can I prevent...contain repeating elements. Knowing how to amplify recombination-prone plasmids and testing your prep can ...recombinases like RecA and RecBCD in bacteria. When two repeating elements love each other very much undergo recombination...recombination often occurs between the long-terminal repeats (LTRs). After recombination, one resulting plasmid...the bacterial selection marker and origin of replication. During bacterial culture, only the plasmid with...the antibiotic resistance gene and origin will replicate, and the transgene will be lost. Over time, bacteria...

...This new system, termed REPAIR (RNA editing for programmable A to I (G) replacement) is the first CRISPR ...RNA editing from the Cas13b REPAIR system To test the robustness of REPAIRv1, the lab targeted 34 pathogenic...transcriptome-wide comparison, REPAIRv2 reduced off-target sites from the 18,385 observed with REPAIRv1 to merely 20. ..., RNA editing doesn't require homology-directed repair (HDR) machinery, and could thus be used in non-... 30-84 nucleotides, and designated this system REPAIRv1. Using next-generation sequencing, they confirmed...HEK293T cells, as assessed using RNA-seq. Since the REPAIRv1 machinery is too large to fit into adeno-associated...identified ADAR2DD(delta984-1090), which decreases the REPAIRv1 construct size from 4,473 bp to 4,152 bp without...

...part of my job, in addition to preparing DNA and generating/replenishing kits, is to find new ways to make...scale DNA preps with multiple samples without cross contamination. We currently use the CosMCPrep system ... throughput DNA prepping by trying out NEB’s new NEBNext Ultra II FS DNA Library Prep Kit for Illumina...of this newly released prep kit. In their prior NEBNext Ultra II DNA Library Prep Kit for Illumina, one... process for library preparation using this kit is: Fragmentation and end repair Adapter Ligation Clean...advancement easier. NEBNext Ultra FS DNA Library Prep Kit To create a DNA library, one must first fragment...mostly used to make new libraries as opposed to replicating them like we do at Addgene), going to the workshop...

...a subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These...dCas-VPR repressor are introduced into the hPSC genome using AAV1 and contain an EGFP reporter. Expression...to provide ready-to-use viral preps. Here are some of the new viral preps from recent months: New GPCR...measurements of norepinephrine. These norepinephrine sensors include GRAB_NE1h (high affinity) and GRAB_NE1m...production in E.coli as simple as following a DNA miniprep protocol!  First, the authors created the pMAK...Expression of the dCas9-KRAB activator or dCas9-VPR repressor in hPSCs is also driven by a doxycycline inducible...promoter. The gRNA piggyBac plasmids include a mRFP reporter. With this system, they could quantify and track...

...validation carried out by the commercial repository. Repositories generally ensure cell line identity and...microbiology, and pharmacology (many of which are represented in Addgene's Special Collections). Arguably, ...invariably much older than those available from biorepositories leading to genotypic and phenotypic artefacts...interpretation in cell culture for this reason. Unreproducible results are rife. Misidentified or cross-contaminated...been known about since 1966 when Stanley Gartler reported that most of the 18 cell lines he had studied ...easier to conduct high quality cell culture. Biorepositories around the world ensure that the cell lines...The oldest, largest, and most diverse of these repositories is the American Type Culture Collection aka ...

...conjugated with a reporter or tag that can then be detected.  Typically, the reporter is an enzyme such...dish and incubate it with a reporter conjugated primary antibody. The reporter reacts with a provided substrate...simplicity we will discuss the more common enzyme-based reporter methods. For enzymatic signal detection, users... sandwich and competitive. In a direct ELISA a reporter enzyme conjugated antibody binds to antigen coated...antibody first binds to the antigen and then a reporter enzyme conjugated secondary antibody binds to ...detection antibody can be directly conjugated with a reporter enzyme or a conjugated secondary antibody can ...).  The sample (purple) is preincubated with a reporter enzyme labeled antibody and then added to the ...

...more efficient than homology-directed repair Homology-directed repair (HDR) stimulated by double strand breaks...during mismatch repair   ✓   ✓ ~ ~ ~ Transiently co-express mismatch repair inhibitor MLH1dn...originally published in Nature, is a “search-and-replace” genome editing technique that mediates targeted...heteroduplex is resolved by the cell’s mismatch repair system, and more recent prime editing innovations...innovations have improved the odds of mismatch repair favoring the edited strand. The first iteration of prime.... We’ll summarize the major ones in Addgene’s repository later in the post. Advantages of prime editing...relatively low, meaning that most Cas9-induced DSBs are repaired by non-homologous end joining. As a result, Cas9...

... Meghan Rego. Reproducible data are key to science, so scientists are used to repeating experiments to...the plasmid used to create the viral prep. In a clean viral prep, like AAV-44362 shown below, more than...confirm their findings. But no scientist wants to repeat an experiment because of poor reagent quality. ...the wrong plasmid? What if bacteria snuck into the prep during the production process? These questions are...contamination can be identified, and we will discard any AAV prep we suspect is contaminated. For AAV-68544, the best...method we wanted to see how low we could go. We prepared a range of spiked samples from 0.1-20% and discovered... detection software confirms the serotype of the prep by interrogating the VGS data for small predetermined...

...biological replicates such as multiple mice in each group and C, technical replicates or repeated measurements...results.  Replicates Replicates are another critical control in biological experiments. Replicates can be...statistical power of an experiment. Technical replicates are repeated measurements of the same sample and are...experimental protocol. In the experiment above, you could repeat the tau western blot several times with the same...difficult to obtain meaningful results.  Biological replicates are distinct samples that are treated identically... on the results of a single mouse. Biological replicates help you to determine which responses arise from...will be used (Burry 2011). It does not need to be repeated for each experimental run as long as the protocol...

...fluorescent reporter, directly couple silencing of your gene target to silencing of the reporter. This is...target sequence against the fluorescent reporter in an inverted repeat orientation. Therefore, a single hairpin...Figure 1). The plant kingdom has expanded its repertoire of Dicer enzymes and these different Dicer-like...desired target of RNAi is expressed as inverted repeats separated by a loop region that facilitates the...conclusion was based upon numerous phenotype discrepancies between gene silencing and knockout methods ... complementary to your gene target as inverted repeats (Figure 1). With this technique, multiple genes...fusing your gene target sequence to a fluorescent reporter sequence (discussed below). Examples of these ...

...their relative representation in the edited pool. The black vertical dotted line represents the cut site...vertical black dotted line represents the cut site. Cutting and error-prone repair typically result in mixed...also provides a Knockout score (KO-score) which represents the proportion of cells that have either a frameshift...For a multiplex sample, ICE includes visual representations of all detected edit types in the sample. The...complete, a new screen appears with a graphical representation of the results and a list of the analyzed samples...used. Currently, ICE is configured for Cas9 fromStreptococcus pyogenes (SpCas9). ICE Score - The editing...correlation coefficient (r) is also computed and reported.  The higher the R2 value, the more confident ...

...suggested roles in transcriptional repression, activity dependent de-repression, chromatin remodeling, gene ...Programmable RNA Editing. Cell Reports 32:107878 . https://doi.org/10.1016/j.celrep.2020.107878 Sinnett SE, ...focused on therapeutic strategies that use gene replacement therapy, gene editing, MECP2 reactivation, RNA...RNA editing, RNA trans-splicing, and protein replacement to reverse the impacts of this disease.  As a...is a strong translational candidate for gene replacement therapy. In 2014, RSRT brought together a strong...have shown exciting and promising results for repairing mutations in models of neurological disease. Klaus... wild-type human iPSCs and then to successfully repair the mutation (Le et al., 2019). Beam Therapeutics...

...Fluorecent Proteins in Nano Lanterns Reporter gene assays A reporter gene, such as luciferase, usually serves... is also the most commonly used bioluminescent reporter. This beetle emits a yellow-green light with a...several studies utilized luciferase as a genetic reporter in plant and mammalian cells. Luciferase assays...available to choose from) is now a common feature in reporter plasmids.  Learn How Luciferase Can Be Used In...transcription within cells, where detection of the reporter protein or its enzymatic activity is measured....expression can be determined by detection of the reporter in a specific assay, which ideally would have .... Production of photons by this bioluminescent reporter occurs slower than fluorescent-based methods, ...

...have newly-available AAV preparations added this year. We’ve added new viral preps of the dopamine sensor...2022.   Find iGluSnFR3 plasmids here! Find viral prep here: pAAV.hSyn.FLEX.iGluSnFR3.v857.PDGFR.codonopt... sustained deep-tissue imaging in vivo. Image reproduced from Liu et al. 2022, under CC-BY-NC license....license.   Find JEDI-2P plasmids here! Find viral preps here: pAAV-EF1a-DIO-JEDI-2P-WPRE and pAAV-EF1a-DIO-JEDI...activity of specific RTKs are limited. In a new preprint, Jared Toettcher and colleagues describe pYtags...of interest and co-expressing a cytosolic ZtSH2 reporter, they observe fluorescence localizes to the plasma...And orthogonal sensors with different ITAM/SH2 reporter pairs allow multiplexed measurement of different...

...must undergo homology-directed repair (HDR), a distinct cellular DNA repair pathway. Accomplishing this ...biallelic modifications. Most commonly, DSBs are repaired by the nonhomologous end-joining (NHEJ) pathway... convenient for generating gene knockouts, NHEJ repair does not allow introduction of specific sequence...involves simultaneously introducing a homologous DNA repair template, such as a sin­gle-stranded oligodeoxynucleotide... is relatively rare and DSBs are predominantly repaired by NHEJ. While other groups have focused on strategies...the same allele, probably due to concomitant NHEJ repair. At the PSEN1 locus, over 90% of the HDR edited...one Step 2 – Edit the cells a second time using a repair template that corrects the blocking mutation and...

... and DNA repair. Black arrows indicate natural cellular processes. BER = base excision repair. Note: hypoxanthine...subsequently converted to thymine through DNA replication or repair. Fusing an inhibitor of uracil DNA glycosylase...Homology-Directed Repair Learn about base editing for plants Read about four base editor reporters Additional...the low editing efficiency of homology-directed repair (HDR), the pathway for resolving DNA double-strand...glycosylase (UGI) to dCas9 prevents base excision repair, which would change the U back to a C base. To increase...newly synthesized” to the cell. Thus, the cell repairs the DNA using the U-containing strand as a template...uracil N-glycosylase (UNG) during base excision repair. Adding a second copy of the UNG inhibitor, UGI...