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Optogenetics + CRISPR, Using Light to Control Genome Editing

Posted by Caroline LaManna on Mar 8, 2016 10:30:00 AM

Scientists around the world have been making major improvements to CRISPR technology since its initial applications for genome engineering in 2012. (Check out our CRISPR 101 eBook for everything you need to know about CRISPR.) Like CRISPR, optogenetics has also been making headlines over the past decade. Optogenetics uses genetically encoded tools, such as microbial opsins, to control cellular activities using light. In 2015, scientists combined CRISPR and optogenetics techniques to develop a variety of photoactivatable CRISPR tools. These tools allow scientists to use light to externally control the location, timing, and reversibility of the genome editing process. Read on to learn about the various light-controlled CRISPR tools available to researchers - some readily found at Addgene.

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Topics: Optogenetics, CRISPR

CRISPR Methods for Bacterial Genome Engineering

Posted by Mary Gearing on Mar 3, 2016 10:30:00 AM

This post was updated on Dec 5, 2017.

Although CRISPR systems were first discovered in bacteria, most CRISPR-based genome engineering has taken place in other organisms. In many bacteria, unlike other organisms, CRISPR-induced double stranded breaks are lethal because the non-homologous end-joining (NHEJ) repair pathway is not very robust. In many cases, homology-directed repair does not function effectively either, but scientists have devised means of co-opting phage genetic systems to facilitate homologous recombination in bacteria. These quirks change the way CRISPR-mediated genome engineering functions in bacteria, but have no fear - plasmids from Addgene depositors are making it easier than ever to do CRISPR editing in E. coli and other commonly-used bacterial species. Read on to learn about the tools available for bacteria and some of the applications for which they’ve been used.

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Topics: Genome Engineering, CRISPR

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

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

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