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CRISPR 101: Epigenetics and Editing the Epigenome

Posted by Mary Gearing on Jun 24, 2020 1:45:00 PM

Originally published Feb 14, 2017 and updated Jun 24, 2020.

Epigenetic modifications are an additional layer of control over gene expression that go beyond genomic sequence. Dysregulation of the epigenome (the sum of epigenetic modifications across the genome) has been implicated in disease states, and targeting the epigenome may make certain processes, like cellular reprogramming of iPSCs, more efficient. In general, epigenetic chromatin modifications are correlated with alterations in gene expression, but causality and mechanisms remain unclear. Today, targeted epigenetic modification at specific genomic loci is possible using CRISPR, and Addgene has a number of tools for this purpose.

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Topics: CRISPR, CRISPR 101

CRISPR 101: Ribonucleoprotein (RNP) delivery

Posted by Andrew Hempstead on Sep 6, 2018 8:02:59 AM

CRISPR has greatly enhanced the ability of scientists to make genomic alterations, bringing about a revolution in genome engineering, with new techniques rapidly being developed. Performing a CRISPR experiment requires delivery of, at minimum, two components: the Cas9 protein and a guide RNA (gRNA) targeting your genomic site of interest. This is commonly performed by transfecting cells with a plasmid, such as PX459, which encodes Cas9 and contains a site for inserting a custom gRNA.  While this methodology has proven to be incredibly valuable to scientists, there are some potential complications that must be considered when using this method:

  1.     Cells must be amenable to transfection or viral transduction
  2.     Appropriate promoters must be chosen for both Cas9 and gRNA expression  
  3.     Plasmid DNA may be incorporated into the genome
  4.     Off-target effects can occur due to prolonged Cas9 expression
  5.     The requirement for Cas9 transcription and translation delays editing
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Topics: CRISPR, CRISPR 101, CRISPR Expression Systems and Delivery Methods

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!

Download a PDF version of the CRISPR Cheat Sheet here!

 

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Topics: CRISPR, CRISPR 101

CRISPR 101: Cas9 Nickase Design and Homology Directed Repair

Posted by Mary Gearing on Mar 15, 2018 8:59:40 AM

By mutating one of two Cas9 nuclease domains, researchers created the CRISPR nickase. Nickases create a single-strand rather than a double-strand break, and when used with two adjacent gRNAs, can lower the probability of off-target editing. But that’s not all! New research from IDT (Integrated DNA Technologies) has shown that a nickase approach can improve homology directed repair (HDR) rates, provided you follow some simple design rules described below.

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Topics: CRISPR, CRISPR 101, Cas Proteins

CRISPR 101: RNA Editing with Cas13 and REPAIR

Posted by Mary Gearing on Nov 30, 2017 9:01:02 AM

Cas13 enzymes are quickly becoming major players in the CRISPR field. Just a year after Abudayyeh et al. (2016) identified Cas13a (C2c2) as a RNA-targeting CRISPR enzyme, Cox et al. have adapted Cas13b for precise RNA editing. This new system, termed REPAIR (RNA editing for programmable A to I (G) replacement) is the first CRISPR tool for RNA editing, and it displays high specificity and targeting flexibility. We’ll walk through how this tool was developed and potential ways you can use it in your research.

Find the plasmids from Cox et al. here!

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Topics: CRISPR, CRISPR 101, Cas Proteins

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