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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

Plasmids 101: Monitoring Cell Mobility Using Fluorescent Proteins

Posted by Benoit Giquel on Aug 15, 2017 9:24:39 AM

In complex metazoans, rapid cell division and large scale cell mobility are essential processes during embryonic development. These are required for a growing organism to make the complicated transition from a clump of cells to a fully differentiated body. In contrast, these dynamic processes are largely absent in adult organisms, where tissues structures are more stable and local movements predominate (e.g. a basal progenitor cell migrating to the epithelium). At this stage, only cells from the immune system show wide scale mobility with movement from the bone marrow and other lymphoid organs to specific tissues where they can scan for any signs of danger. In this post we’ll focus on how fluorescent proteins can and have been used to monitor cellular movements in the immune system. The techniques used here could be adapted to studying other systems in which there is large scale cellular movement throughout an organism.

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Topics: Plasmids 101, Fluorescent Proteins

Human Germline Editing Using CRISPR

Posted by Mary Gearing on Aug 10, 2017 10:19:54 AM

Note: After this blog was published, a bioRxiv preprint that questions the conclusion of inter-homologue recombination was released. This blog has not been updated in response to this paper.

Any hint of CRISPR editing in human embryos has been met with a storm of media coverage. But the paper published August 2nd in Nature gives us even more to talk about, as it represents another step towards CRISPR germline editing of disease-causing mutations. But how close are we really, and what new questions does this paper bring up? We’ll sift through the paper to understand what Shoukhrat Mitalipov and his colleagues have achieved, and how the field will move forward from this work.

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Topics: CRISPR

History of Fluorescent Proteins

Posted by A Max Juchheim on Aug 7, 2017 9:58:40 AM

Luminescent molecules are very useful tools because we can easily detect and measure the light they emit. Proteins that give off light include chemiluminescent proteins, like luciferases, as well as fluorescent ones, like Green Fluorescent Protein (GFP). These molecules occur naturally in bioluminescent organisms, but their real power lies in the clever ways sceintists have adapted them for use in the laboratory.

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Topics: Fluorescent Proteins

Plasmids 101: An Inside Look at NGS Plasmid Quality Control

Posted by Amanda Hazen on Aug 3, 2017 8:49:45 AM

All plasmids coming through Addgene’s doors are now verified by next-generation sequencing (NGS) to provide you with more data. For most plasmids, we are now able to confirm the full insert and backbone sequence. In order to manage and interpret these data, we’ve updated our quality control workflow.  Here’s a peek at how our process has improved now that we’re backed by the power of NGS!

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Topics: Inside Addgene, Plasmids 101

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