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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

PITChing MMEJ as an Alternative Route for Gene Editing

Posted by Mary Gearing on Feb 23, 2016 10:30:00 AM

If you follow CRISPR research, you know all about using non-homologous end-joining (NHEJ) to make deletions or homology-directed repair (HDR) to create precise genome edits. But have you heard of another double-stranded break repair mechanism: MMEJ (microhomology-mediated end-joining)? MMEJ, a form of alternative end-joining, requires only very small homology regions (5-25 bp) for repair, making it easier to construct targeting vectors. Addgene depositor Takashi Yamamoto’s lab has harnessed MMEJ to create a new method for CRISPR gene knock-in, termed PITCh (Precise Integration into Target Chromosomes). Using their PITCh plasmids, GFP knock-in cell lines can be created in about a month and a half, without the need for complicated cloning of homology arms.

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Topics: CRISPR, CRISPR Protocols and Tips, Other CRISPR Tools

CRISPR Protocol for Genomic Deletions in Mammalian Cell Lines [Video]

Posted by Guest Blogger on Feb 18, 2015 10:09:22 AM

The following post was contributed by Daniel Bauer and Matthew Canver of Boston Children’s Hospital and Harvard Medical School. Addgene is proud to present a video reprint of the CRISPR article "Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9" from the Journal of Visualized Experiments (JOVE). The video publication by Stuart Orkin and Daniel Bauer's labs details the use of CRISPR/Cas9 to create genomic deletions in mammalian cell lines. Below Bauer and Canver discuss the motivations behind this research.

 

Using CRISPR/Cas9 for targeted genomic deletions

We were inspired to produce intrachromosomal deletions based on the experiments of Kim and colleagues using zinc finger nucleases to harness non-homologous end joining repair (NHEJ) [1]. Our initial work was with TALENs, in collaboration with the Porteus lab [2]. With the advent of CRISPR/Cas9, we began to explore the paired double-strand break (DSB) approach at a variety of loci. We were pleasantly surprised by the efficiency of the method. One observation was an inverse relationship between deletion size and frequency [3].

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Topics: CRISPR, CRISPR Protocols and Tips

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