Recombinase-based State Machines Enable Order-dependent Logic in vivo

Posted by Guest Blogger on Jul 28, 2016 10:30:00 AM

This post was contributed by guest blogger Nathaniel Roquet, a PhD student in the Harvard Biophysics program and researcher in the Lu Lab at MIT.

Note: The following blog post reduces the content of our paper, “Synthetic recombinase-based state machines in living cells” (1), into a more straight-forward, concise explanation of how to adapt our engineered devices, recombinase-based state machines for your own experimental needs. For more context, exposition, and detail, please refer to the paper.

Why Might One Be Interested in State Machine Technology?

Biological research has produced a massive amount of information regarding which regulatory proteins, signaling molecules, mutations, and environmental conditions drive certain cellular behaviors, but little is known about the order or timing of these factors. Recombinase-based state machines (RSMs), which take on a particular DNA-sequence configuration (state) based on the identity and order of a particular set of inputs, may be used to better understand and engineer cellular processes that are influenced by temporally ordered biochemical events.

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Topics: Genome Engineering, Synthetic Biology

Cpf1 Update: Comparison to Cas9 and NgAgo

Posted by Mary Gearing on Jul 14, 2016 10:30:00 AM

In 2015, Feng Zhang’s lab characterized two Cpf1 nucleases, distant cousins of well-known Cas9. Cpf1 cleaves DNA in a staggered pattern and requires only one RNA rather than the two (tracrRNA and crRNA) needed by Cas9 for cleavage. Now, two new studies show that Cpf1 displays lower off-target editing than Cas9, confirming that this protein is well suited for genome editing. 

Find Cpf1 Plasmids at Addgene

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Topics: Genome Engineering, CRISPR

Generating Mouse Models Using CRISPR/Cas9

Posted by Guest Blogger on Jul 12, 2016 10:30:00 AM

This post was contributed by guest bloggers, Wenning Qin and Haoyi Wang.

CRISPR/Cas9 is revolutionizing the mouse gene-targeting field. Mice have long been extremely useful in the lab – they are relatively small and easy to work with, making them the go-to choice for studying mammalian biology. Similar to any model, mice are not without their problems, but much genetic and physiological data have been accumulated over the years using them. Indeed, the future of mouse work is bright as it is now easier than ever to manipulate the mouse genome using CRISPR/Cas9.

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Topics: Genome Engineering, CRISPR

Tips for CRISPR Gene Editing in Mice

Posted by Guest Blogger on Jun 28, 2016 6:59:27 AM

This post was contributed by guest blogger Samantha Young.

The use of CRISPR/Cas9 for gene editing has expanded since its adaptation for use in mammalian cells in 2012-2013. Researchers are now using this system in ever more creative ways, (Wang et al., 2013, Cho et al., 2014). There are several variants of the CRISPR/Cas9 system floating around, and many pre-designed plasmids containing these variants ready for purchase. But what is the easiest and fastest way to use the system in mice? We'll have a post that goes into the mouse genome editing process in a bit more detail in the coming weeks, but, in this post, we will outline a simple method for selecting the guide RNA, validating its efficacy in vitro, and using it in mouse embryos to generate gene modified mouse lines. Hopefully this post will help get your in vivo research up and running as soon as possible!

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Topics: Plasmid Technology, Genome Engineering, Lab Tips, CRISPR

Comparing Cas9 to NgAgo: Can the Argonautes Best CRISPR?

Posted by Mary Gearing on Jun 9, 2016 10:30:00 AM

Biologists are going gaga over the newest gene-editing protein - a DNA-cleaving Argonaute from Natronobacterium gregoryi, or NgAgo for short. Addgene has already distributed this plasmid all over the world, and the question on everyone’s minds is: could NgAgo replace CRISPR? Such a drastic shift won’t happen overnight, but there are a few reasons why you might choose NgAgo over CRISPR proteins Cas9 or Cpf1 - keep reading to learn more!

Find New Tips in the Han Lab's NgAgo Protocol Here!

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Topics: Plasmid Technology, Genome Engineering, CRISPR

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