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Cas9 Activators: A Practical Guide

Posted by Guest Blogger on Aug 18, 2016 10:30:00 AM

This post was contributed by guest bloggers Marcelle Tuttle and Alex Chavez, researchers at the Wyss Institute for Biologically Inspired Engineering.

Listen to Our Podcast Interview the Alex Chavez

Background on Cas9 Activators


CRISPR/Cas9
is an enormously plastic tool and has taken the scientific world by storm. While Cas9 has been most widely used to create specific edits in DNA, there has also been significant work on constructing Cas9 transcriptional activators. These constructs allow for the upregulation of essentially any gene by fusing mutants of Cas9 deficient in DNA cutting activity to a transcriptional activation domain (Fig 1).

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Topics: CRISPR

CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

Posted by Guest Blogger on Aug 9, 2016 10:30:00 AM

This post was contributed by guest blogger, Aneesh Karve, CTO at Qult Data. This post was originally published on the Quilt Genomics Blog and is republished here with permission.

Quilt is a collaborative database for genomics. In this article, Quilt CTO Aneesh Karve, shows how to design experiments that work anywhere in the genome. Aneesh's research interests include proteomics, machine learning, and visualization for big biology.
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Topics: CRISPR

Google Forums Round Up: First Impressions of NgAgo

Posted by Guest Blogger on Aug 4, 2016 10:30:00 AM

Update (November 18, 2016): Researchers from a variety of institutions recently reported their inability to recapitulate the results of Gao et al 2016 in a letter to Protein & Cell.

Update (August 3rd, 2017) THE ORIGINAL NgAgo ARTICLE DISCUSSED IN THIS POST HAS BEEN RETRACTED AND FOLLOW UP STUDIES HAVE FAILED TO DEMONSTRATE GENOME EDITING BY THIS TOOL

This post was contributed by guest blogger Pooran Dewari. Any views in this post are those of the guest blogger and do not necessarily represent the views of Addgene. Addgene performs Sanger sequencing on select regions of all distributed plasmids as part of quality control, but does not perform functional tests.

The newest genome engineer sharing the stage with much-lauded CRISPR-Cas9 is DNA-guided endonuclease NgAgo! We'll discuss how NgAgo is faring with users in a minute, but, to start, let's review why NgAgo is in the spotlight and take a moment to remember that NgAgo has only been available for genome editing for a few months. More time is required for its optimization and development before it can truly be pitted against CRISPR head-to-head. 

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Topics: Genome Engineering, CRISPR

Site Directed Mutagenesis by PCR

Posted by Guest Blogger on Aug 2, 2016 10:30:00 AM

This post was contributed by guest blogger, Kristian Laursen from Cornell University.

Site directed mutagenesis is a highly versatile technique that can be used to introduce specific nucleotide substitutions (or deletions) in a tailored manner. The approach can be used in conventional cloning (to introduce or remove restriction sites), in mapping of regulatory elements (to mutate promoters/enhancers in reporter constructs), in functional analysis of proteins (to perform alanine scanning mutagenesis or targeted substitution of key residues), and in SNP analysis (to introduce naturally occuring SNPs in a plasmid context). The technique is also highly relevant in this age of CRISPR; site-directed mutagenesis generally applies to plasmids, but may also facilitate genome editing. Tailored mutations are commonly introduced to endogeneous DNA through homology-directed repair (HDR) of a CRISPR/Cas9 induced double-stranded break. This site-directed genome editing requires a template of high homology to the endogenous target, yet to facilitate the repair, the template should be resistant to Cas9 cleavage. If a plasmid contains the template, site-directed mutagenesis can be used to mutate the PAM sequence (an NGG sequence critical for Cas9 cleavage), thereby rendering the resulting construct resistant to Cas9 induced cleavage.

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Topics: Lab Tips, Protocols

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

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