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Andrew Hempstead

Andrew Hempstead is a Senior Scientist at Addgene. Andrew's interests include genome engineering and microbiology.

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Isolating Genomic Regions of Interest with the CRISPR System

Posted by Andrew Hempstead on May 30, 2019 8:44:22 AM

While much of CRISPR research has focused on genome editing, numerous discoveries have been made using the Cas9 nuclease in the absence of genomic alterations. These studies utilize a catalytically inactive form of Cas9 known as dCas9 (Jinek et al., 2012). Like Cas9, dCas9 can bind to a specific DNA sequence via a targeting gRNA. But dCas9 does not cleave the DNA. Much of the research using dCas9 has focused on transcriptional activation using a fusion to a transcriptional activator such as VP64 (Gilbert et al., 2013), or repression of transcription through binding a promoter region to inhibit association of transcriptional activators (Qi et al., 2013). However, the fusion of dCas9 with a protein tag allows for the isolation of a genomic region of interest targeted by a gRNA.

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Topics: CRISPR

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

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