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Hassle-free 96-well format epitope tagging using Cas9 ribonucleoprotein

Posted by Guest Blogger on Jun 28, 2018 11:01:10 AM

This post was contributed by guest blogger Pooran Dewari, a postdoc in Steve Pollard’s lab at the MRC Centre for Regenerative Medicine (CRM), Edinburgh.

Most commercial antibodies do not work in pull-down assays: Epitope tagging provides a solution

Proteins - the workhorses of the cell – never work alone in the cellular milieu. It is, therefore, critical to understand how proteins interact with one another (or with DNA) to perform diverse biochemical tasks in the cell. One of the most popular approaches to study protein interactions is the pull-down assay, wherein a protein of interest can be captured along with its associated partners. Common pull-down assays include immunoprecipitation mass spectrometry (IP/MS) and chromatin immunoprecipitation (ChIP). In IP/MS, a target protein is first immunoprecipitated - along with its associated protein complexes - from the cell-lysate using antibodies against the target protein. The captured protein complexes are then analysed by mass spectrometry to identify the interacting proteins. Similarly, in ChIP-seq assays, chromatin fragments that are bound by a protein of interest are pulled-down and later coupled to high-throughput sequencing to identify genome-wide binding patterns of the target protein.

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Topics: CRISPR, Techniques

CRISPR Cheat Sheet

Posted by Tyler Ford on May 31, 2018 10:43:15 AM

At Addgene we periodically have Science Clubs where we present developments in biology research to the whole company with the goal of educating both scientists and nonscientists alike. As part of these presentations, we generally create one page cheat sheets that attendees can use to quickly reference information that they (hopefully) learn at science club. In this post you'll find our CRISPR Cheat Sheet from @megearing's recent science club presentation about genome editing and CRISPR. We hope you find this cheat sheet useful! 

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Topics: Genome Engineering, CRISPR

Analyzing CRISPR Editing Results with ICE from Synthego

Posted by Guest Blogger on May 8, 2018 9:00:20 AM

This article was contributed by Jessica Roginsky, Scientific Support Lead at Synthego. Article source: Step-by-Step Guide for Analyzing CRISPR Editing Results with ICE on Synthego’s blog.

CRISPR-based genome engineering has revolutionized the gene editing field by making experimental workflows considerably easier, faster, and more efficient than previous methods. Still, generating reliable results from CRISPR edit data requires the help of robust software tools. As a consequence, a critical step in the gene editing workflow - analyzing the data - is often under-appreciated or over-looked. 

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Topics: CRISPR

Cas13d: Small RNA-targeting CRISPR enzymes for transcriptome engineering

Posted by Mary Gearing on May 3, 2018 9:48:09 AM

RNA-editing Cas13 enzymes have taken the CRISPR world by storm. Like RNA interference, these enzymes can knock down RNA without altering the genome, but Cas13s have higher on-target specificity. New work from Konermann et al. and Yan et al. describes new Cas13d enzymes that average only 2.8 kb in size and are easy to package in low-capacity vectors! These small, but mighty type VI-D enzymes are the latest tools in the transcriptome engineering toolbox.

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Topics: CRISPR

Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

Posted by Mary Gearing on Apr 5, 2018 8:42:28 AM

CRISPR genome editing has made it easier to create knockout alleles in a variety of species, including the standard laboratory mouse. It’s also made targeted insertions relatively simple in C. elegans and bacteria. But CRISPRing typical mouse models, including creating Cre-dependent conditional alleles, has remained a challenge. Enter Easi-CRISPR: a method that harnesses the power of ssDNA donor molecules for homology directed repair. Using long ssDNA donors, the Gurumurthy and Ohtsuka groups have obtained an average knock-in efficiency of 30-60%. This is much more favorable than previous methods yielding 1-10% knock-in. Read on to learn how you can make CRISPR mouse model generation easi-er!

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Topics: CRISPR, Cre-lox

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