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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: Plasmid Technology, Genome Engineering, CRISPR, Techniques

CRISPR 101: Multiplex expression of gRNAs

Posted by Mary Gearing on Jan 28, 2016 10:50:00 AM

This post was updated on Dec 5, 2017.

CRISPR makes it easy to target multiple loci - a concept called multiplexing. Since CRISPR is such a robust system, editing or labeling efficiency doesn’t usually change when you add multiple gRNAs. Sound good? Addgene has many tools to help you multiplex - we’ll use mammalian plasmids to introduce you to some of your potential options and cloning methods, but please scroll down for plasmids suitable for other model systems, including E. coli, plants, Drosophila, and zebrafish!

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Topics: Genome Engineering, CRISPR, CRISPR 101, Plasmid Kits

Enhancing CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9

Posted by Tyler Ford on Dec 16, 2015 10:30:00 AM

As evidenced by all the CRISPR publications, press, and plasmids out there, it’s obvious that CRISPR is a ground-breaking technology that’s already had a huge impact on research and will be affecting our everyday lives very soon. Not only is CRISPR having effects on various biological disciplines, the base technology itself is constantly improving. Cas9 variants have been modified for genome editing, activating gene expression, visualizing genomic loci, and much more. Now, researchers from the Zhang, Joung, and Doudna labs have improved the on-target specificity of the Cas9 nuclease with engineered variants: eSpCas9SpCas9-HF1, & HypaCas9.

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Topics: Genome Engineering, CRISPR

The PAM Requirement and Expanding CRISPR Beyond SpCas9

Posted by Joel McDade on Nov 12, 2015 10:30:00 AM

This post was updated on Dec 5, 2017.

Diverse genomes and genomic targets require a variety of tools to engineer them effectively. Since the discovery and engineering of dCas9, the CRISPR toolbox has expanded to include a variety of natural and engineered Cas proteins. Read on to learn how these tools can be used to expand CRISPR's reach to new genomic loci. 

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Topics: Genome Engineering, CRISPR

The CRISPR Software Matchmaker: A New Tool for Choosing the Best CRISPR Software for Your Needs

Posted by Guest Blogger on Nov 3, 2015 10:30:00 AM

This post was contributed by guest blogger Cameron MacPherson at the Institut Pasteur

CRISPR Software and the Piñata Effect

Two years ago I was a part of a group (Biology of Host-parasite Interactions, Institut Pasteur, Paris) that changed genome editing in the malaria community for the better (Nat. Biotechnol., 2014). Given the timing, it shouldn’t be a surprise that the CRISPR system was involved. Today, that same laboratory enjoys a successful edit rate of over 90% in their work editing the genome of Plasmodium falciparum (the parasite that causes malaria). I attribute their success to technical expertise, thoughtful single guide RNA (sgRNA) design, and the abnormally low GC content of the Plasmodium falciparum genome. To put this last point into perspective, the Plasmodium falciparum genome contains only 0.66 million targetable NGG PAM sites whereas the human genome has about 300 million. With such a sparsely targetable genome, off-targeting is less of a worry and on-targeting likely more efficient. 

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Topics: Genome Engineering, CRISPR

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