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CRISPR Protocol for Genomic Deletions in Mammalian Cell Lines [Video]

Posted by Guest Blogger on Feb 18, 2015 10:09:22 AM

The following post was contributed by Daniel Bauer and Matthew Canver of Boston Children’s Hospital and Harvard Medical School. Addgene is proud to present a video reprint of the CRISPR article "Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9" from the Journal of Visualized Experiments (JOVE). The video publication by Stuart Orkin and Daniel Bauer's labs details the use of CRISPR/Cas9 to create genomic deletions in mammalian cell lines. Below Bauer and Canver discuss the motivations behind this research.

 

Using CRISPR/Cas9 for Targeted Genomic Deletions

We were inspired to produce intrachromosomal deletions based on the experiments of Kim and colleagues using zinc finger nucleases to harness non-homologous end joining repair (NHEJ) [1]. Our initial work was with TALENs, in collaboration with the Porteus lab [2]. With the advent of CRISPR/Cas9, we began to explore the paired double-strand break (DSB) approach at a variety of loci. We were pleasantly surprised by the efficiency of the method. One observation was an inverse relationship between deletion size and frequency [3].

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Topics: Genome Engineering, Lab Tips, CRISPR, Protocols

An “elegans” Approach to Better CRISPR/Cas9 Editing Efficiency

Posted by Guest Blogger on Jan 27, 2015 10:13:47 AM

This post was contributed by Jordan Ward who is a postdoctoral fellow at UCSF.

Emerging CRISPR/Cas9 editing technologies have transformed the palette of experiments possible in a wide range of organisms and cell lines. In C. elegans, one of the model organisms which I use to study gene regulation during developmental processes, CRISPR/Cas9 allows us to knock out sequences and introduce mutations and epitopes with unprecedented ease. In the last year, several advances in C. elegans genome editing using CRISPR/Cas9 have emerged, which I will describe below. These new C. elegans approaches rapidly enrich for editing events without the need for any selective marker to remain in the edited animal. To my knowledge these approaches have not yet been extended to other organisms/cell lines, though it is likely that many aspects will broadly improve editing efficiency.

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Topics: Plasmid Technology, Genome Engineering, CRISPR

7 Simple Strategies to Resolve Conflicts with Difficult Supervisors

Posted by Guest Blogger on Jan 21, 2015 8:30:00 AM

This post was contributed by Dora Farkas of www.FinishYourThesis.com and is one of many posts in our career series. Click here to subscribe to our career posts.

“The only healthy communication style is assertive communication.” -  Jim Rohn, Author, Entrepreneur, Motivational Speaker

Do you have a difficult supervisor, or do you have to work with difficult people? Each time I ask this question at my workshops, I get nods from nearly every participant.

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Topics: Career

Resume Writing for Non-academic Science Careers

Posted by Guest Blogger on Dec 18, 2014 11:06:00 AM

This post was contributed by Theresa Liao of the University of British Columbia.

When transitioning from an academic science career path to a non-academic one, one of the biggest changes (and perhaps challenges) is the need to present yourself using a resume. Indeed, instead of having all the pages in a Curricula Vitae to showcase your publications, academic performances, and research experiences, you now have merely two pages to convince your potential employer that you are the right candidate for the job.

How can you incorporate the skills developed during graduate school into your resume? How can you stand out among all the candidates applying for the position?

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Topics: Career, Science Communication, Career Readiness

Choosing Your Fluorescent Proteins for Multi-Color Imaging

Posted by Guest Blogger on Oct 9, 2014 11:00:00 AM

This post was contributed by Kurt Thorn of the Nikon Imaging Center at UCSF.

A common requirement for live cell imaging experiments is the ability to follow multiple fluorescently tagged species simultaneously. To do so with fluorescent protein labels requires multiple fluorescent proteins whose excitation and emission spectra differ sufficiently for them to be imaged in distinct fluorescent channels on the microscope. With the proliferation of fluorescent proteins in recent years, there are many fluorescent protein combinations that can be imaged together, but this also means that the choice of fluorescent proteins requires some thought.

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Topics: Lab Tips, Fluorescent Proteins

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