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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, CRISPR Protocols and Tips

Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

Posted by Leila Haery on Mar 7, 2017 10:30:00 AM

We all know that in the lab there are often little tricks that are essential for experiments but that nobody talks about. After months of troubleshooting, those people who did not tell you that essential thing ask incredulously, “You seriously didn’t add 3 microliters of 5 mM star anise?” This is something I was expecting when I set out to make my first CRISPR/Cas9 gene edit. I wanted to inactivate the gene BRAF (a kinase implicated in several human cancers) in A549 cells (a human lung cancer cell line), armed only with viruses obtained through Addgene’s viral service and the methods sections of scientific articles (gasp). To my delight, not only was I able to make the edits without any reagent-grade endangered Martian chicory root, but considering this is a needle in a haystack type of objective, it was surprisingly easy. It’s true, I CRISPRed. In this post, I’ll summarize the basic steps and analyses, and give what I think are the main tips for each step of performing and analyzing a gene edit using Addgene’s lentiviral CRISPR tools.

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Topics: CRISPR, CRISPR Protocols and Tips

Sequencing Options for CRISPR Genotyping

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

This post was contributed by guest blogger Søren Hough, the Head Science Writer at Desktop Genetics.

One of the most important steps in the CRISPR experimental process is validating edits. Regardless of which CRISPR genome editing system you use, there remains a chance that the observed phenotype was caused by an off-target mutation and not an edit in the target gene.

The validation process, also known as CRISPR genotyping, is critical to demonstrating causal relationships between genotype and assayed phenotype. Verifying these connections can help alleviate the reproducibility crisis in biology. It is key to address these concerns as CRISPR use grows across the life sciences and to establish standardized validation techniques for academia, industry, and especially the clinic.

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Topics: CRISPR, CRISPR Protocols and Tips

CRISPR/Cas9 FAQs Answered!

Posted by Caroline LaManna on Mar 13, 2014 12:08:00 PM

As Kendall mentioned in Tuesday's blog post, keeping up with the newest CRISPR technologies and their applications can be exhausting. A quick search for "CRISPR", short-hand for Clustered Regularly Interspaced Short Palindromic Repeats, in Pubmed returned 728 articles (3/12/2014). With so many options for CRISPR plasmid tools and numerous experimental design decisions to make, it makes sense that scientists, many of whom are venturing into genome editing for the first time, have lots of questions.

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Topics: CRISPR, CRISPR Protocols and Tips

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