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We narrowed to 790 results for: Cre

Showing: 121 - 130 of 790 results

  1. xCas9: Engineering a CRISPR Variant with PAM Flexibility

    Type
    Blog Post
    Published
    March 28, 2018, 6:52 p.m.
    ...genome-wide off-targets, since increasing PAM flexibility might also increase off-target activity. Surprisingly...gone one step further, using directed evolution to create xCas9, an enzyme recognizing a broad range of PAMs...PAMs like NG, GAA, and GAT, but also displaying increased editing specificity. We’re excited to learn more...easy, right? Yes and no. If you’re just trying to create a knockout, you might be happy targeting anywhere...precise edits or knock-ins. Editing efficiency decreases drastically once your cut site is more than 10...insertion site. Base editing, another method to create point mutations, also has a strict editing window... PAM sequences and/or broader PAM flexibility. Creating xCas9 through directed evolution Hu et al. used...

  2. CRISPR 101: Cytosine Transversion Editors

    Type
    Blog Post
    Published
    March 25, 2025, 1:15 p.m.
    ...nucleobase component of the nucleoside inosine. Created with BioRender.com. Although it was a huge ...it finds a PAM, Cas9 unwinds the adjacent DNA to create a region of separate strands called an R-loop (...of the enzyme. Figure 2: CRISPR R-loop. Created with BioRender.com. Base editors take advantage...activity window restricted to the stretch of ssDNA created by Cas9 binding. Pro tip! In CRISPR, the non-target...BER process was excising the incorrect U base, creating an “abasic,” or empty, DNA site, and then replacing...in the same journal on the same day.) This team created a similar construct that they called a Glycosylase...GBE, for its reliance on the glycosylase UNG to crease abasic sites (Zhao et al., 2021). Their GBE produced...

  3. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    Published
    Oct. 27, 2020, 1:15 p.m.
    ...ones? Here, I’ll cover some screening methods to identify them. Visual screening Regardless of the RNAi-trigger...endogenous miRNA precursors can be engineered to created artificial miRNAs. The processed miRNA or siRNAs...conclusion was based upon numerous phenotype discrepancies between gene silencing and knockout methods ...RNAi in the context of a high throughput survival screen found that either technology was equivalently precise... can be divided into three fundamental steps: creation of the RNAi trigger delivering the RNAi trigger...the first stage of an RNAi-based investigation. Creation of the RNAi trigger requires a few initial considerations...these constructs include pGAPi, which allows easy creation of long hpRNA with Gateway-based cloning and straightforward...

  4. Targeted Mutagenesis with EvolvR

    Type
    Blog Post
    Published
    Feb. 21, 2019, 1:15 p.m.
    ...modular nature, the Dueber Lab created a few versions of EvolvR. To increase the mutation rate of EvolvR,...bacteriophage T7 DNA polymerase increased the processivity of PolI3M, and thereby increasing the editing window of...forward genetics harnesses this at a large scale by screening diverse libraries of genetic variants. Several...nucleotides. Both the Bassik Lab and the Chang Lab have created methods that introduce diversification by targeting... integration of oligonucleotide libraries of a discrete size at the target site. The use of such libraries...occasionally introducing mutations (red nucleotide). Created with BioRender.com. The initial version of ...per generation of wild-type E. coli, while only increasing the standard mutation rate seen during DNA replication...

  5. Supporting Rare Disease Research with the 2020 BeHEARD Awards

    Type
    Blog Post
    Published
    Sept. 8, 2020, 2 p.m.
    ... Association for Creatine Deficiencies: Compensating for SLC6A8 mutations in Creatine Transporter Deficiency...SLC6A8 gene, also known as the creatine transporter. The Association for Creatine Deficiencies (ACD) is a parent-let...regeneration of ATP from ADP is creatine. In order to reach the brain, creatine needs to be transported from...Deficiency Creatine Transporter Deficiency (CTD) is a rare X-linked inherited neurometabolic condition and... and one of three Cerebral Creatine Deficiency Syndromes (CCDS) that present as global developmental delays... blood and cross the blood brain barrier using creatine transporters. However, mutations in the transporter...SLC6A8 impede this transport and result in global creatine deficiency, which is characterized by development...

  6. Viral Vectors 101: Preparing Pooled Libraries

    Type
    Blog Post
    Published
    July 13, 2023, 1:15 p.m.
    ...libraries here at Addgene, including cDNA, barcoding, screening, and gRNA libraries. Most of the pooled libraries...determined, larger lentiviral preps can be used for screening-scale transduction of cells, resulting in cell...library. These cells can then be used for genetic screening, followed by sequencing and further data analysis...some of your plasmids didn’t amplify after your screen is completed!  Producing Your Lentiviral Library... any issues.  Keep in mind that a low MOI will increase the chances of a cell being infected by only one... one viral particle, but it also increases the chances that a cell doesn’t get infected at all. To make...as seeding density and reagent concentration.  Screening time Phew! You are now (finally!) ready to use...

  7. Hot Plasmids - December 2019 - CRISPR pooled library, LipoGlo, CRISPRa, and Voltage Imaging Tools

    Type
    Blog Post
    Published
    Dec. 10, 2019, 1:58 p.m.
    ...pooled libraries have been created in lentiviral systems for mammalian screens, which enables researchers... as the screen readout. This retroviral mouse CRISPR pooled library can be used for screening of cells...pooled libraries are popular tools for genetic screens, enabling genome-wide interrogation of the genes...required for cellular processes. For a successful screen, it is necessary to identify the gRNAs that elicit...are poorly transduced by lentivirus, and thus screening with available CRISPR libraries was not possible...solve this issue, Sarah Teichmann and colleagues created a retroviral CRISPR knockout library using gRNAs... from mice that constitutively express Cas9 and screen for genes that affect Th2 differentiation and activation...

  8. How to Negotiate a Successful Lab Start Up Budget

    Type
    Blog Post
    Published
    Sept. 1, 2015, 2:30 p.m.
    ...goals and the resources. Instead of creating a start up budget, create a start up list.  In this post we...award by giving you step by step instructions on creating a budget, making decisions based on that budget...budget, and negotiating in regards to the budget. Creating a budget We start by using your research specific...budgetary needs from your experimental goals. When creating your budget, you should take the following steps... that timeline Avoid writing down prices as you create your list. This helps to ensure that all of the...”, “colony”, etc. This will help save time by screening out irrelevant candidates, and, more importantly...doesn’t have to be complicated, or difficult to create this list. Start BEFORE searching for a faculty...

  9. Stabilized Bacterial Promoters: Constant Gene Expression at any Copy Number

    Type
    Blog Post
    Published
    Sept. 4, 2018, 12:53 p.m.
    ...gene expression. By itself, increasing the copy number of a gene increases its expression. However, if...as plasmid copy number increases, the amount of repressor protein also increases thereby intensifying repression...When they increased concentrations of IPTG, a constitutive promoter upstream of GFP increased expression...that is responsive to a repressor protein, the increase in expression due to copy number is kept in check...origins of replication, the stabilized promoters created by Segall-Shapiro et al. showed nearly no position-dependent...stationary phase, the number of plasmids per cell increases 4-5 fold (for pUC plasmids) or ~2 fold (for p15a...compared to a constitutive promoter. To simulate the increase in plasmid copy number during the transition to...

  10. Design Tips for Prime Editing

    Type
    Blog Post
    Published
    Jan. 23, 2025, 2:15 p.m.
    ... edited. Created with BioRender.com. Add silent mutations near point mutations to create 3-base (...target can increase the odds of incorrect editing outcomes, especially when MMR is inhibited. Created with ...edit. Figure 1: Prime Editing schematic. Created with BioRender.com. The pegRNA specifies both...PBS) and reverse transcriptase template (RTT). Created with BioRender.com. The first base of the ...to Cas9 with canonical sgRNA structure (right). Created with BioRender.com. What to edit The sequence... mismatched bases can often evade MMR (right). Created with BioRender.com. For specific prime editing...nicking sgRNAs ~50 bp from the prime editing nick. Created with BioRender.com. PE3b and PE5b: In this...
Showing: 121 - 130 of 790 results