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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

A Match Made in Heaven: CRISPR and AAV

Posted by Mary Gearing on Jul 14, 2015 10:30:00 AM

This post was updated on Dec 4, 2017.

CRISPR genome editing has quickly become the most popular system for in vitro and germline genome editing, but in vivo gene editing approaches have been limited by problems with Cas9 delivery. Adeno-associated viral vectors (AAV) are commonly used for in vivo gene delivery due to their low immunogenicity and range of serotypes allowing preferential infection of certain tissues. However, packaging Streptococcus pyogenes (SpCas9) and a chimeric sgRNA together (~4.2 kb) into an AAV vector is challenging due to the low packaging capacity of AAV (~4.5 kb.) While this approach has been proven feasible, it leaves little room for additional regulatory elements. Feng Zhang's group previously packaged Cas9 and multiple gRNAs into separate AAV vectors, increasing overall packaging capacity but necessitating purification and co-infection of two AAVs.

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Topics: CRISPR, Viral Vectors

Even more elegant: Single injection CRISPR/Cas9 in C. elegans

Posted by Mary Gearing on Jul 7, 2015 11:36:00 AM

In the summer of 2013, a remarkable nine papers describing CRISPR/Cas9 genome engineering methods for C. elegans were released, signaling a new era in C. elegans research. Homology directed repair (HDR), which enables insertion of custom genomic modifications, is very robust in C. elegans, and the methods for HDR-mediated modification continue to be improved. New work from Bob Goldstein’s lab at the University of North Carolina has made CRISPR in C. elegans even easier - now, one can generate a fluorescent protein fusion, transcriptional reporter, and loss-of-function allele in just one injection step! The entire protocol takes about 2-3 weeks but requires less than eight hours worth of hands-on time.

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Another Pathway into Cells: iTOP

Posted by Mary Gearing on Jun 23, 2015 4:37:00 PM

Primary cells recapitulate the natural biology of a cell type of interest better than immortalized lines derived from the same cell type; however, their usage has been limited by technical problems. For instance, it’s much more difficult to introduce a gene of interest into primary cells, so most primary cell lines require viral infection. A new paper from Niels Geijsen’s lab suggests that primary cells may be better transduced using only protein. Read on for a description of the lab’s iTOP protein-only transduction method and its potential applications to CRISPR/Cas9 genome editing.

 

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Topics: CRISPR, Techniques

Easing the Protein Purification Process with pCri

Posted by Mary Gearing on Jun 19, 2015 11:08:00 AM

Protein purification can be one of the most stressful lab activities. Working with proteins requires a substantial amount of properly folded, relatively pure protein, but getting to this stage is often much easier said than done. As reviewed in our Plasmids 101 series, proteins are overexpressed from a plasmid construct, most often in special E. coli strains designed for protein expression. Cultures are then lysed and the protein of interest is purified using an affinity tag. Additional tags may be used to improve protein stability and solubility.

Determining the best way to express one’s protein of interest can save a lot of time later.

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Topics: Hot Plasmids, Plasmid Kits

Science Communication Snapshot: DayCon 2015

Posted by Mary Gearing on Jun 17, 2015 5:04:11 PM


SITN DayCon 2015 Team

Here at Addgene, we’re dedicated to advancing and sharing science! In association with the Harvard graduate student organization Science in the News (SITN), we recently sponsored a first-time event called DayCon. DayCon is a one-day conference aimed at the general public that provides an accessible introduction to various scientific topics. Over twenty graduate student volunteers worked hard to make this Saturday event a success, a true testament to the commitment of SITN members.

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Topics: Scientific Sharing, Science Communication

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