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Searchable and Sortable gRNAs for Your Next CRISPR Experiment

Posted by Nicole Waxmonsky on May 17, 2016 10:30:00 AM

This post was written jointly by Addgenies Brook Pyhtila and Nicole Waxmonsky

Resource sharing shortens the time needed to go from planning an experiment to performing one.  At Addgene, over 120 labs have deposited CRISPR reagents, including many gRNA-containing plasmids (McDade et al, 2016). Many of the gRNAs contained within these plasmids have been used successfully in peer reviewed articles. If you’re targeting your favorite gene with CRISPR, using one of these validated gRNAs can save you the time that would be spent making and testing entirely new gRNA designs. You can now easily find many validated gRNAs in our newly curated Validated gRNA Target Sequence Table.

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Topics: Genome Engineering, Inside Addgene, CRISPR

Plasmids 101: Colony PCR

Posted by Guest Blogger on May 12, 2016 10:30:00 AM

This post was contributed by guest blogger Beth Kenkel, a Research Assistant in the Department of Pediatrics at the University of Iowa. If you're interested in guest blogging, let us know!

Molecular cloning requires some method of screening colonies for the presence of an insert. Traditionally this has been done with restriction enzyme digest; however colony PCR can accomplish the same thing in less time and for less money. The key steps to colony PCR are: 1) design primers to detect the presence of your insert; 2) set up a standard PCR reaction (primers, dNTPs, polymerase) using the supernatant of lysed bacteria as template; and 3) run your PCR product on a gel to analyze product size. This blog post discusses some of the key things to consider when performing colony PCR.

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Topics: Plasmid How To, Plasmids 101, Protocols, Plasmid Cloning

Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?

Posted by Mary Gearing on May 10, 2016 10:30:00 AM

Over 25 million people worldwide are currently infected with the
lentivirus HIV-1. Today, HIV-1 can be controlled with antiviral therapies such that the virus is undetectable in the blood. But the virus doesn’t completely disappear; it just hides in latently infected cells. To truly cure HIV-1, researchers need to vanquish these hidden viral reservoirs, and CRISPR may be the way to accomplish this tough job! Kamel Khalili’s lab at Temple University has demonstrated two potential strategies for CRISPR-HIV therapeutics - one using dCas9-SAM to activate HIV-1 transcription and destroy infected cells, the other using wild-type Cas9 to remove the HIV-1 genome from infected cells. Read on to learn how CRISPR can take on HIV-1 in vitro, and what obstacles must be overcome for clinical success.

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Topics: Genome Engineering, CRISPR

The Challenges of Cell Culture

Posted by Guest Blogger on May 5, 2016 10:30:00 AM

This post was contributed by ATCC Outreach Scientist Nick Amiss.

Cell Culture in the Present Day

Cell culture is a widespread tool used in the fields of oncology, virology, immunology, microbiology, and pharmacology (many of which are represented in Addgene's Special Collections). Arguably, oncology has benefited most as cancer cells are amenable to culture in vitro. Virologists too have benefited from the ability to propagate viruses in cell culture; if there were no cells to infect, there would be no viable viruses to study. Interestingly, due to certain high profile problems surrounding cell culture over the last few decades some pharmacologists have tended to avoid cell culture in favour of biochemical assays followed by in vivo testing. The mantra being “you can’t trust those whacky cell lines”. This may certainly have been a valid concern for the discipline (or lack thereof) in times gone past but these days it’s never been easier to conduct high quality work in cell culture.

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Topics: Lab Tips

CRISPR Antimicrobials

Posted by Mary Gearing on May 3, 2016 10:30:00 AM

The crisis of antibiotic resistance is upon us, and the world is unprepared. Each year in the United States, two million people will be infected by antibiotic resistant bacteria. Even when researchers develop new antibiotics, the onset of resistance is swift, as few as five years after introduction. Current antibiotic strategies are nonspecific - they harm any bacterial cell without a resistance gene, allowing resistant bacteria to multiply, spreading their resistance genes throughout the bacterial population. But what if we could specifically target only virulent or antibiotic resistant bacteria with a weapon that they’ll have less potential to become resistant to? CRISPR may provide a method for doing just that. While challenges remain in the delivery of these agents, CRISPR antimicrobials could become our newest line of defense against bacteria.

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Topics: Genome Engineering, CRISPR

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