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CRISPR 101: CRISPR-mediated Plant Base Editors

Posted by Guest Blogger on Jan 3, 2019 8:35:29 AM

This post was contributed by Kutubuddin Molla, a Fulbright Visiting Scholar at the Pennsylvania State University.

Imagine you are dealing with a defective gene, Xm, the sequence of which is identical to the correct gene, Xw, except for a single base. If you heard about CRISPR, one question probably comes to mind: can CRISPR be applied to fix the defective base precisely?

Until 2016, precise single base changes were only possible through exploiting the homology-directed repair (HDR) pathway which occurs in cells at low frequencies and relies on the efficient delivery of donor DNA to facilitate repair. Since the development of CRISPR-mediated base editing (BE), these types of repairs can now be done more efficiently than before. A base editor precisely changes a single base with an efficiency typically ranging from 25-75%, while the success of precise change via HDR limited to 0-5%. This blog post covers a brief review of different basic BE technologies and their adaptation for plant genome editing.
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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 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

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