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3D Printing Meets CRISPR Cas9

Posted by Guest Blogger on Oct 17, 2017 8:55:24 AM

This post was contributed by Tim Herman, director of the MSOE center for BioMolecular Modeling and the CEO of 3-D Molecular Designs.

Have you ever held your favorite protein in the palm of our hand? Well, actually – have you ever held a model of your favorite protein in your hand? At 3D Molecular Designs, we create physical models of proteins using 3D printing technology. The tagline for our company is “…where molecules become real”.  We sometimes forget that the models we create are around 30 million times larger than the real thing. Nevertheless, the models are compelling, and I encourage you to hold one.

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Topics: CRISPR

Fluorescent Tagging of Endogenous Genes with SapTrap

Posted by Michelle Cronin on Oct 12, 2017 10:26:13 AM

Since the discovery of GFP over 50 years ago, the growing spectrum of fluorescent proteins (FPs) has been an invaluable resource for studying the organization and function of cellular systems. FPs have been used to track protein localization, cell structure, intracellular trafficking, and protein turnover rates. Additionally, by engineering FP fusions associated with cellular organelles, scientists have been able to study many cellular processes, including mitosis, mitochondrial fission/fusion, nuclear import, and neuronal trafficking. Although FPs have enabled discovery of many cellular mechanisms, there are some limitations to working with FPs. Overexpression of fluorescently tagged proteins can lead to improper protein localization, protein aggregation, or disruption of normal protein function, and ultimately misinterpretation of the protein’s cellular role.

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Topics: CRISPR, Fluorescent Proteins

CRISPR 101: Targeting RNA with Cas13a (C2c2)

Posted by Joel McDade on Sep 21, 2017 10:07:21 AM

CRISPR, and specifically Cas9 from S. Pyogenes (SpCas9), is truly an exceptional genome engineering tool. It is easy to use, functional in most species, and has many application (see a review of CRISPR applications here). That said, SpCas9 is not the only game in town, and several non-SpCas9 molecules have been characterized. Early research suggests that these molecules may circumvent the limitations associated with SpCas9 (see our blog entitled “Which Cas9 do I choose for my experiment”). A novel protein, Cas13a (previously referred to as C2c2), has several unique properties that make it particularly useful and further expand the CRISPR toolbox. This blog post will cover how Cas13a was identified, the structure and function of Cas13a with a focus on what makes this molecule unique, and the various applications of Cas13a.

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Topics: CRISPR, CRISPR 101

Some Like it Hot: Thermostable GeoCas9

Posted by Beth Kenkel on Sep 14, 2017 8:40:16 AM

Cas9 is the genome editing tool of choice for a number of model organisms: mammalian cells, yeast, drosophila, plants, worms, zebrafish, frogs, some bacteria; but not thermophilic (high heat loving) bacteria. Until recently the only available Cas9 proteins were isolated from mesophilic (medium heat loving) bacteria, such as Streptococcus pyogene’s SpCas9. These Cas9 proteins don’t work well at high temperatures, so to use them in thermophiles, bacteria must be grown at lower temps. This approach only works for facultative thermophiles (high OR medium heat loving), but not obligate thermophiles. However, the recent discovery of GeoCas9 by the Doudna lab has opened up the field of thermophilic bacteria to CRISPR/Cas9 genome editing.

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Topics: Genome Engineering, CRISPR

3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

Posted by Guest Blogger on Sep 5, 2017 9:58:42 AM

This post was contributed by guest bloggers Dominik Paquet and Dylan Kwart from Ludwig-Maximilians-University in Munich and Marc Tessier-Lavigne’s lab at the Rockefeller University in NYC.

The CRISPR/Cas9 system is a versatile tool for precise gene editing in many organisms and model systems. We have used CRISPR/Cas9 extensively for the purpose of making sequence-specific changes in human induced pluripotent stem cells (iPSCs). The CRISPR/Cas9 com­plex is very efficient at introducing double stranded breaks (DSBs) into genomic DNA in many cell types and often results in biallelic modifications. Most commonly, DSBs are repaired by the nonhomologous end-joining (NHEJ) pathway, leading to nonspecific nucleotide insertions, dele­tions or other mutations, referred to as ‘indels’. While this is convenient for generating gene knockouts, NHEJ repair does not allow introduction of specific sequence changes.

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Topics: CRISPR

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