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Finding nucleic acids with SHERLOCK and DETECTR

Posted by Alyssa Cecchetelli on Aug 30, 2018 8:28:06 AM

Sensitive and specific nucleic acid detection is crucial for clinical diagnostics, genotyping, and biotechnological advancements. Current methods of nucleic acid detection however, either lack the sensitivity or the specificity to detect nucleic acids at low concentrations and/or are too expensive, time-consuming, and complex to use outside of standard laboratories. Recently scientists have utilized CRISPR-Cas9 protein variants, Cas13, and Cas12a, to develop simple, portable, and inexpensive platforms to reliably detect nucleic acids at the atomolar level.

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Topics: CRISPR, Cas Proteins, Other CRISPR Tools

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, Other CRISPR Tools

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, Other CRISPR Tools

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, Other CRISPR Tools

CAPTURE-ing Chromatin Interactions: Using CRISPR-dCas9 to Study Gene Regulation

Posted by Beth Kenkel on Dec 7, 2017 9:16:43 AM

Plasmids can be amazing and simple tools for studying gene regulation. They are used to study how transcription factors and other trans-regulatory elements (TREs) and some cis-regulatory elements (CREs), like promoters, influence gene expression. However, scientists frequently return to native chromosomes because chromatin context matters. The impact of TREs and CREs on gene expression is commonly investigated via Chromatin Immunoprecipitation (ChIP) and chromatin capture techniques, respectively, but these two separate methods are not without their own technical challenges. Enter the Xu Lab's CAPTURE, a method for identifying TREs and CREs that partners CRISPR’s targeting abilities with the strength of the biotin-streptavidin interaction. CAPTURE is capable of identifying old and new TREs and CREs, CRE-CRE interactions, and has even provided enough data for Liu et al to re-draw the beta-globin locus regulation model. Read on to learn more about this captivating tool!

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Topics: CRISPR, Other CRISPR Tools

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