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Beth Kenkel

Beth Kenkel is currently a research scientist in the Department of Laboratory Medicine at the University of Washington. She is particularly interested in science communication and in vitro diagnostics. Follow Beth on twitter @ElizabethKenkel.

Recent Posts

Anti-CRISPRs: Switching Off CRISPR-Cas9

Posted by Beth Kenkel on May 23, 2017 10:30:00 AM

CRISPR-Cas9 technology is constantly evolving. Variants of Cas9 can be used for genome editingactivating gene expression, repressing gene expression, and much more. But there’s one thing that’s been missing: a way to shut off Cas9’s activity after it’s been turned on. The concern is that the longer Cas9 remains active in a cell, the greater chances there are for off-target edits to occur. Although methods to switch on Cas9 activity using light or drugs have been developed, the field lacked an “off-switch” for Cas9.

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Topics: CRISPR

Multiplex Genome Editing with CRISPR-Cpf1

Posted by Beth Kenkel on May 9, 2017 10:12:15 AM

There’s a new development for CRISPR-Cpf1 genome editing!  A recent paper from Feng Zhang's lab describes how to use Cpf1 for multiplex genome editing.  For a few reasons, Cpf1 is a simplified system for editing multiple targets compared to Cas9.  Read on to learn more about Cpf1 multiplexing.  For an in-depth review of Cpf1, check out this blog post or see Addgene's CRISPR guide page for a review of Cas9.  For a brief comparison of Cpf1 vs. Cas9, see the table below.

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Topics: CRISPR

Rosella: A Fluorescent pH-Biosensor for Studying Autophagy

Posted by Beth Kenkel on Apr 13, 2017 10:30:00 AM

Rosella is a pH-sensitive fluorescent biosensor that was recently deposited with Addgene by Dr. Mark Prescott. This system was developed for monitoring and analyzing autophagy of cytosol and organelles in yeast cells. Autophagy (Greek for “self-eating”) is induced by a lack of nutrients and targets cytosol and organelles to the vacuole/lysosome for degradation and recycling. The key to Rosella’s autophagy-sensing abilities is that its fluorescence emission spectra changes when it goes from a more neutral pH compartment, ­­like the cytosol, to the higher pH of the vacuole. Read on to learn more about prior methods for studying autophagy and how Rosella improves upon them.

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Topics: Fluorescent Proteins

Deep Mutational Scanning with One Pot Saturation Mutagenesis

Posted by Beth Kenkel on Feb 22, 2017 10:30:00 AM

Scientists use deep mutational scanning to simultaneously test how multiple amino acid changes affect a protein of interest’s function. This technique relies on the generation of a plasmid library that expresses all desired variants of a protein. Applying a selective pressure winnows the pool down to plasmids expressing variants with optimal function. High-throughput DNA sequencing is then used to measure the frequency of each variant during the selection process. Each variant is assigned a functional score based on its library frequency before selection compared to its library frequency after selection. Key to this process is the ability to generate full libraries of mutant proteins. Researchers from the Whitehead lab developed One pot saturation mutagenesis as a quick and easy technique that can be used to generate complex libraries of mutant plasmids ready for deep mutational scanning.

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Topics: Techniques

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

Posted by Beth Kenkel on Dec 15, 2016 10:57:02 AM

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

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