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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

6 Steps to Submitting a Resume That Gets Seen

Posted by Joanne Kamens on Jul 9, 2015 10:30:00 AM

Expanding your network of relationships early and often is the most effective tactic a scientist in training can adopt to ensure opportunities in the future. Studies show that the majority of job offers arise as a result of existing professional (and personal) relationships. However, most job seekers will and should apply for jobs posted online as one part of their job search. This is especially true of scientists seeking a first job after the academic bench. Utilizing existing relationships as part of the job application process can dramatically increase the chances of being seriously considered for an open position. 

Check out Joanne's Reddit AMA

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Topics: Career

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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Walking beside Academia and giving it a high five: My transition from graduate work to a position at Addgene

Posted by Tyler Ford on Jul 2, 2015 4:37:00 PM

Just over a month ago I finished up my PhD in Biological and Biomedical Sciences at Harvard University and entered a new role here at Addgene as an Outreach Scientist. I used to spend my days (and often my nights :D) engineering E. coli to produce biofuels in Pamela Silver’s Lab (plasmid page here). Now I spend much less time wrangling bacteria and instead help Addgene fulfill its mission of helping scientists share information and, of course, plasmids. My primary duties are to manage this very blog (you’ll have to let me know how I’m doing a few months down the line!) and to visit scientists in person to figure out ways we can make Addgene better and make scientists’ lives easier.

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Topics: Career

Sleeping Beauty Awakens for Genome Engineering

Posted by Emma Markham on Jun 30, 2015 10:00:00 AM

Transposons are sequences of DNA that can move around in a genome. In a laboratory setting, transposons can be used to both introduce genes into an organism’s genome (see figure) and to disrupt endogenous genes at the site of insertion. In both of these cases, transposons combine the advantages of viruses and naked DNA while eliminating some of the drawbacks. Specifically, viruses are able to infect and replicate in host cells, but they are susceptible to cells’ defense mechanisms. The use of non-viral vectors, like transposons, avoids many, though not all, of these defenses. For some applications of genome engineering - such as certain forms of gene therapy - avoiding the use of viruses is also important for social and regulatory reasons.

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Topics: Plasmid Technology, Genome Engineering

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