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CRISPR-Cas9: Tips for Optimizing sgRNA Activity

Posted by Guest Blogger on Feb 19, 2016 10:18:31 AM

 This post was contributed by John Doench of the Broad Institute.

For more infomation on gRNA design, see our post: How to Design Your gRNA for CRISPR Genome Editing

Whether designing a small number of sgRNAs for a gene of interest, or an entire library of sgRNAs to cover a genome, the ease of programing the CRISPR system presents an embarrassment of riches of potential sgRNAs. How to decide between them? By taking into account both on-target efficacy and the potential for off-target activity, experiments utilizing CRISPR technology can provide a straightforward means of determining loss-of-function phenotypes for any gene of interest.

Predicting sgRNA Efficacy

We have recently examined sequence features that enhance on-target activity of sgRNAs by creating all possible sgRNAs for a panel of genes and assessing, by flow cytometry, which sequences led to complete protein knockout (1).

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Topics: Plasmid Technology, CRISPR

28 Hot Plasmid Technologies from 2015

Posted by Tyler Ford on Dec 23, 2015 10:30:00 AM



At Addgene we're continually impressed with the amazing plasmid technologies developed by our community of depositors. With over 40,000 plasmids avaliable in the repository, we can't give all of them attention they righlty deserve, but, in this post, we'll provide a small sampling of the many amazing new plasmid tools that have come through our doors in 2015. Do you have a favorite new plasmid tool from the past year? Let us know about it in the comments or shoot us an e-mail at blog@addgene.org and maybe we can write a blog post about it!

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Topics: Plasmid Technology, Hot Plasmids, Plasmid Kits

Teaching an Old DOG New Tricks: Controlling Protein Activity with GFP

Posted by Mary Gearing on Nov 24, 2015 10:30:00 AM

At Addgene, we love GFP, and we’re always excited when depositors find new ways to make this workhorse protein even more useful! From FPs optimized for oxidizing environments to photoconvertible variants, it seems like GFP is always learning new things. Now, work from Connie Cepko’s lab allow researchers to activate transcription or Cre recombinase activity only in the presence of GFP. These systems, known as T-DDOG and Cre-DOG, respectively, repurpose popular GFP reporter lines for more sophisticated experimental manipulations, saving the time and money needed to develop new lines.

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Topics: Plasmid Technology, Synthetic Biology, Fluorescent Proteins, Cre-lox

Tips for Screening with Yeast Two Hybrid Systems

Posted by Jessica Welch on Oct 22, 2015 10:30:00 AM

Two hybrid systems were developed in Saccharomyces cerevisiae in 1989 and are still used extensively to screen for molecular interactions in the cell, including protein-protein, protein-DNA and protein-RNA interactions.

The 1980s saw a flurry of discovery in the field of eukaryotic transcriptional activation and cell biology. During this period, proteins were successfully expressed as fusions that retain their individual activities (1). Researchers also discovered the modular format of some transcriptional activators: that the DNA binding domain (DBD) and transcriptional activation (TA) domains retain their individual activities when separated (2), and that DBD and TAs from different systems could be combined effectively (3).

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Topics: Plasmid Technology, Yeast

pSiM24: Simplifying Plant Genetic Engineering

Posted by Mary Gearing on Sep 29, 2015 10:30:00 AM

As previous blogs have noted, plants are an important foundation for life on Earth. Selective breeding methods have shaped the plants that we grow and eat, and genetic engineering will continue to improve plant nutrition, yield, and pest resistance. Much of plant genetic engineering revolves around Agrobacterium tumifaciens. Agrobacterium carries a “tumor-inducing” or Ti plasmid, which allows it to transfer genetic material into the host plant genome. Scientists have worked to optimize this system for gene transfer, studying the stability of modified Ti plasmids during plant infection, as well as plasmid yield during preparation in E. coli. Addgene depositor Indu Maiti has created a new and versatile binary Ti vector for both transient and stable gene expression applications in plants. This smaller, easily customizable vector functions in multiple species, including tobacco and Arabidopsis.

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Topics: Plasmid Technology, Plant Biology

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