Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

Posted by Guest Blogger on Dec 15, 2016 10:57:02 AM

This post was contributed by guest blogger, Beth Kenkel, a research scientist at the University of Washington.

Restriction enzyme cloning is the workhorse of molecular cloning; however, one of its biggest limitations is that sequence modifications can only be made at restriction enzyme cut sites. The lambda red system is an alternative method that can be used for cloning or genome engineering and is based on homologous recombination. It allows for direct modification of DNA within E. coli and is independent of restriction sites. The lambda red system is derived from the lambda red bacteriophage and its use as a genetic engineering tool is frequently called recombineering - short for homologous recombination-mediated genetic engineering.  It can be used to make an assortment of modifications: insertion and deletion of selectable and non-selectable sequences, point mutations or other small base pair changes, and the addition of protein tags. It also has the flexibility to modify the E. coli chromosome, plasmid DNA or BAC DNA. 

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Topics: Genome Engineering, Techniques, Microbiology

Plasmids 101: Knockout/Knock-In Plasmids

Posted by Benoit Giquel on Dec 1, 2016 10:30:00 AM

One of the most powerful strategies to investigate a gene's function is to inactivate, or "knockout", the gene by replacing it or disrupting it with an piece of DNA designed in the lab. Specially constructed plasmids can be used to replace genes in yeast, mice, or Drosophila through homologous recombination. The concept is simple: deliver a template with a modified version of the targeted sequence to the cell which will recombine the template with the endogenous gene. Here, we'll describe the techniques and the plasmids used to inactivate specific genes in mammalian cells. Despite the popularity of CRISPR-based knockout/knock-in systems, these systems remain valuable, especially in cases where CRISPR cannot be used (e.g. there are no suitable PAM sequences nearby or your gene of interest is difficult to target specifically with a gRNA). Be sure to keep these techniques in mind when choosing a knockout strategy!

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Topics: Plasmid How To, Genome Engineering, Plasmids 101

Single Base Editing with CRISPR

Posted by Mary Gearing on Aug 16, 2016 10:30:00 AM

When we talk about CRISPR applications, one negative always comes up: the low editing efficiency of homology-directed repair (HDR). Compared to the random process of non-homologous end joining, HDR occurs at a relatively low frequency, and in nondividing cells, this pathway is further downregulated. Like all CRISPR applications that use wild-type Cas9, editing by HDR also has some potential for off-target cleavage even when gRNAs are well designed. Rather than try to improve HDR, Addgene depositor David Liu’s lab created new Cas9 fusion proteins that act as “single base editors.” These fusions contain dCas9 or Cas9 nickase and the rat cytidine deaminase APOBEC1, which can convert cytosine to uracil without cutting DNA. Uracil is subsequently converted to thymine through DNA replication or repair. Komor et al. estimate that hundreds of genetic diseases could be good targets for base editing therapy, not to mention the potential basic and preclinical research applications. Read on to learn about this new way to make point mutations using CRISPR without double-stranded breaks.

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Topics: Genome Engineering, 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.

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

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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