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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

Call for Guest Bloggers

Posted by Tyler Ford on Mar 2, 2016 10:00:00 AM

Whenever possible, we love to give scientists the opportunity to share their knowledge. One of our goals is to be a go-to source of information on recent advances in the biological sciences and techniques that simplify and expedite research. We recognize, however, that we can’t do it all ourselves. Therefore, in this blog post we’re taking a moment to reach out to you, our readers, and ask you to share your expertise through our blog.

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Topics: Career, Inside Addgene

Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

Posted by Brook Pyhtila on Mar 1, 2016 10:30:00 AM

Over the past decade, scientists have developed and fine tuned many different ways to clone DNA fragments which have provided appealing alternatives to restriction enzyme cloning. These newer technologies have become more and more common, and for good reason. They offer many advantages over the traditional restriction enzyme cloning we once relied exclusively on. In this blog post, I will go over some advantages, disadvantages, and examples of how scientists are using Gibson assembly to put together DNA fragments.

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Topics: Plasmid Technology, Plasmids 101, Protocols, Plasmid Cloning

Experimenting with New Careers while in Grad School

Posted by Guest Blogger on Feb 25, 2016 10:30:00 AM

Thie post was contributed by guest blogger Melanie Fox, founder and executive director of Central Indiana Science Outreach and a Postdoc at Indiana University School of Medicine.

It’s not always easy to figure out what you want to do after graduate school, at least not while you’re still in the thick of it. About three or four years into my PhD in Molecular Biology, I realized I wanted a career in science outreach: engaging the public to promote an awareness and understanding of science. Like most science PhD programs, mine was geared toward careers in academia or industry. Luckily, I discovered that there are many ways to get a taste of a variety of careers while still working towards your degree. For me, experimenting with the career and networking opportunities available to me as a graduate student culminated in my founding a nonprofit called Central Indiana Science Outreach, or CINSO. We organize fun science events for adults and professional development opportunities for researchers interested in connecting with the public. My experiences in grad school, though often called “nontraditional,” helped prepare me to start CINSO. Here, I share some of the tips I learned along the way and hope they’ll help you make the most of the opportunities you’re provided throughout your PhD program.

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Topics: Career, Career Readiness

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

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