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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

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, Genome Engineering, Plasmid Technology

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, Genome Engineering

Genome Engineering, CRISPR, and Research in Singapore: Interview with Wei Leong Chew

Posted by Tyler Ford on Feb 6, 2018 9:00:56 AM

In today’s podcast, we sit down with Wei Leong Chew, a researcher at the Genome Institute of Singapore who recently started his own lab. We discuss some of the joys and difficulties of getting a lab up and running, and learn a little bit about what it was like for Wei Leong to work in George Church’s lab as a graduate student.

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Topics: Podcast, CRISPR, Genome Engineering

Some Like it Hot: Thermostable GeoCas9

Posted by Beth Kenkel on Sep 14, 2017 8:40:16 AM

Cas9 is the genome editing tool of choice for a number of model organisms: mammalian cells, yeast, drosophila, plants, worms, zebrafish, frogs, some bacteria; but not thermophilic (high heat loving) bacteria. Until recently the only available Cas9 proteins were isolated from mesophilic (medium heat loving) bacteria, such as Streptococcus pyogene’s SpCas9. These Cas9 proteins don’t work well at high temperatures, so to use them in thermophiles, bacteria must be grown at lower temps. This approach only works for facultative thermophiles (high OR medium heat loving), but not obligate thermophiles. However, the recent discovery of GeoCas9 by the Doudna lab has opened up the field of thermophilic bacteria to CRISPR/Cas9 genome editing.

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Topics: CRISPR, Genome Engineering

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