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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 101, CRISPR, Genome Engineering

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 101, CRISPR

New CRISPR Web Resources and #12DaysofCRISPR Recap

Posted by Mary Gearing on Dec 12, 2017 10:13:34 AM

Since the start of the CRISPR revolution, Addgene has distributed over 100,000 CRISPR plasmids. But that’s not our only job - we strive to also give you high-quality educational resources to help you do better research. CRISPR is an incredibly fast-moving field, and we want to make it easy for you to keep up with new developments (and, of course, find plasmids that will be useful to you.)

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Topics: CRISPR 101, CRISPR, Using Addgene's Website

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

CRISPR 101: Targeting RNA with Cas13a (C2c2)

Posted by Joel McDade on Sep 21, 2017 10:07:21 AM

This post was updated on Nov 14, 2017.

CRISPR, and specifically Cas9 from S. pyogenes (SpCas9), is truly an exceptional genome engineering tool. It is easy to use, functional in most species, and has many applications (see a review of CRISPR applications here). That said, SpCas9 is not the only game in town, and other Cas proteins like SaCas9 and Cpf1 can circumvent the limitations associated with SpCas9. A novel protein, Cas13a (previously referred to as C2c2), has several unique properties that further expand the CRISPR toolbox. We'll cover how Cas13a was identified, the structure and function of Cas13a with a focus on what makes this molecule unique, and the various applications of Cas13a.

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Topics: CRISPR, CRISPR 101

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