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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-Cas9: Tips for Optimizing sgRNA Activity

Posted by Guest Blogger on Feb 19, 2016 10:18:31 AM

 This post was contributed by John Doench of the Broad Institute.

For more infomation on gRNA design, see our post: How to Design Your gRNA for CRISPR Genome Editing

Whether designing a small number of sgRNAs for a gene of interest, or an entire library of sgRNAs to cover a genome, the ease of programing the CRISPR system presents an embarrassment of riches of potential sgRNAs. How to decide between them? By taking into account both on-target efficacy and the potential for off-target activity, experiments utilizing CRISPR technology can provide a straightforward means of determining loss-of-function phenotypes for any gene of interest.

Predicting sgRNA Efficacy

We have recently examined sequence features that enhance on-target activity of sgRNAs by creating all possible sgRNAs for a panel of genes and assessing, by flow cytometry, which sequences led to complete protein knockout (1).

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Topics: Plasmid Technology, CRISPR

Plasmids 101: Restriction Cloning

Posted by Tyler Ford on Feb 18, 2016 10:42:06 AM

When cloning by restriction digest and ligation, you use restriction enzymes to cut open a plasmid (backbone) and insert a linear fragment of DNA (insert) that has been cut by compatible restriction enzymes. An enzyme, DNA ligase, then covalently binds the plasmid to the new fragment thereby generating a complete, circular plasmid that can be easily maintained in a variety of biological systems. Read on for an in-depth breakdown of how to do perform restriction digests.

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Topics: Plasmids 101, Protocols, Plasmid Cloning

REPLACR Mutagenesis: Replacing In Vitro Recombination Methods

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

Site-directed mutagenesis (SDM) is one of the key tools researchers use to prove causation in molecular biology and genetics. It can be used to characterize the function of certain regions in a promoter or gene, as well as to study the effects of inactivating/activating mutations. In biomedical research, modeling patient mutations using SDM can help determine if a variant is causal for a given disease. CRISPR has made genomic SDM relatively straightforward, but plasmid-based SDM has lagged behind. While commercial kits are available for making small point mutations, large deletions/insertions require complicated, often costly in vitro assembly methods. A new method, REPLACR-mutagenesis, harnesses the power of bacterial recombineering to create insertions, deletions, and substitutions - at the same efficiency as Gibson Assembly and GeneArt cloning - but at a much lower cost. Read on to find out how to replace your SDM method with REPLACR (Recombineering of Ends of Linearized Plasmids After PCR).

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Topics: Protocols, Techniques, Plasmid Cloning

How to Lead a Great Meeting

Posted by Carissa Fish on Feb 9, 2016 10:30:00 AM

Meetings often get a bad rap as annoying interruptions to our “real” work. However, a well-run meeting can have quite the opposite effect. A great meeting should produce collaboration - a sense of dialogue and community among participants, clarification - new and useful information, and invigoration - a renewed energy for continuing the project after leaving the meeting. Follow the tips below to learn how you can run a top-notch meeting.

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Topics: Career, Inside Addgene, Career Readiness

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