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Mary Gearing

Mary Gearing is a Scientist at Addgene. She got her start as a Science Communications Intern writing for the Addgene blog and website. As a full-time Addgenie, she still enjoys blogging about CRISPR and other cool plasmids!

Recent Posts

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

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

CRISPR Methods for Bacterial Genome Engineering

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

This post was updated on Dec 5, 2017.

Although CRISPR systems were first discovered in bacteria, most CRISPR-based genome engineering has taken place in other organisms. In many bacteria, unlike other organisms, CRISPR-induced double stranded breaks are lethal because the non-homologous end-joining (NHEJ) repair pathway is not very robust. In many cases, homology-directed repair does not function effectively either, but scientists have devised means of co-opting phage genetic systems to facilitate homologous recombination in bacteria. These quirks change the way CRISPR-mediated genome engineering functions in bacteria, but have no fear - plasmids from Addgene depositors are making it easier than ever to do CRISPR editing in E. coli and other commonly-used bacterial species. Read on to learn about the tools available for bacteria and some of the applications for which they’ve been used.

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Topics: Genome Engineering, CRISPR

PITChing MMEJ as an Alternative Route for Gene Editing

Posted by Mary Gearing on Feb 23, 2016 10:30:00 AM

If you follow CRISPR research, you know all about using non-homologous end-joining (NHEJ) to make deletions or homology-directed repair (HDR) to create precise genome edits. But have you heard of another double-stranded break repair mechanism: MMEJ (microhomology-mediated end-joining)? MMEJ, a form of alternative end-joining, requires only very small homology regions (5-25 bp) for repair, making it easier to construct targeting vectors. Addgene depositor Takashi Yamamoto’s lab has harnessed MMEJ to create a new method for CRISPR gene knock-in, termed PITCh (Precise Integration into Target Chromosomes). Using their PITCh plasmids, GFP knock-in cell lines can be created in about a month and a half, without the need for complicated cloning of homology arms.

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Topics: Plasmid Technology, Genome Engineering, CRISPR, Techniques

REPLACR Mutagenesis: Replacing In Vitro Recombination Methods

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

Site-directed mutagenesis (SDM) is one of the key tools researchers use to prove causation in molecular biology and genetics. It can be used to characterize the function of certain regions in a promoter or gene, as well as to study the effects of inactivating/activating mutations. In biomedical research, modeling patient mutations using SDM can help determine if a variant is causal for a given disease. CRISPR has made genomic SDM relatively straightforward, but plasmid-based SDM has lagged behind. While commercial kits are available for making small point mutations, large deletions/insertions require complicated, often costly in vitro assembly methods. A new method, REPLACR-mutagenesis, harnesses the power of bacterial recombineering to create insertions, deletions, and substitutions - at the same efficiency as Gibson Assembly and GeneArt cloning - but at a much lower cost. Read on to find out how to replace your SDM method with REPLACR (Recombineering of Ends of Linearized Plasmids After PCR).

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Topics: Protocols, Techniques, Plasmid Cloning

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