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Overcoming the AAV Size Limitation for CRISPR Delivery

Posted by Mary Gearing on Sep 16, 2020 9:00:00 AM

Originally published Jul 14, 2015 and last updated Sep 16, 2020 by Beth Kenkel. 

CRISPR genome editing has quickly become a popular system for in vitro and germline genome editing, but in vivo gene editing approaches have been limited by problems with Cas9 delivery. Adeno-associated viral vectors (AAV) are commonly used for in vivo gene delivery due to their low immunogenicity and range of serotypes allowing preferential infection of certain tissues. However, packaging Streptococcus pyogenes (SpCas9) and a gRNA together (~4.2 kb) into an AAV vector is challenging due to its packaging capacity of AAV (~4.7 kb). While this approach has been proven feasible, it leaves little room for additional regulatory elements. Feng Zhang's group previously packaged Cas9 and multiple gRNAs into separate AAV vectors, increasing overall packaging capacity but necessitating purification and co-infection of two AAVs.

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Topics: CRISPR, CRISPR Expression Systems and Delivery Methods

Plasmids 101: Screening Strategies Used in Plasmid Cloning

Posted by Jennifer Tsang on Sep 15, 2020 9:15:00 AM

If you’re cloning a plasmid, you’ll need a way to find the needle in the haystack: the one perfect clone that contains the plasmid you’re looking for out of the many cells that don’t. One way to begin the search is by using selection strategies, where only cells that have gained or lost a specific gene survive (ex: antibiotic resistance marker). In a previous blog post, we covered how to use positive and negative selection in plasmid cloning

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Topics: Plasmids 101, Plasmid Cloning, Plasmids

CRISPR 101: Multiplex Expression of gRNAs

Posted by Mary Gearing on Sep 10, 2020 7:45:00 AM

Originally published Jan 28, 2016 and last updated Sep 10, 2020 by Jennifer Tsang.

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 on one plasmid. 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: CRISPR, CRISPR 101, CRISPR gRNAs

Supporting Rare Disease Research with the 2020 BeHEARD Awards

Posted by Jennifer Tsang on Sep 8, 2020 10:00:00 AM

The Rare Genomics Institute BeHEARD (Helping Empower and Accelerate Research Discoveries) Challenge provides grants for rare disease research. This year is our fourth year working with the Rare Genomics Institute by providing plasmid-based tools for rare disease research.

Congratulations to the winners! Below, we highlight their research and how they will use plasmids and viral preps from Addgene to further their work.

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Topics: Addgene News, Awards

Optogenetics + CRISPR, Using Light to Control Genome Editing

Posted by Caroline LaManna on Sep 3, 2020 8:15:00 AM

Originally published Mar 8, 2016 and last updated Sept 3, 2020 by Nyla Naim.

Scientists around the world have been making major improvements to CRISPR technology since its initial applications for genome engineering in 2012.  Many of these advances have stemmed from the goal of reducing off-target Cas9 activity. The use of nickases, prime editing, anti-CRISPR proteins, and other techniques all aim to improve targeting specificity or reduce the duration of Cas9 activity. 

The field of optogenetics is renowned for enabling precise temporal and spatial control. Optogenetics uses genetically encoded tools, such as microbial opsins, to control cellular activities using light. In 2015, scientists combined CRISPR and optogenetic 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, Other CRISPR Tools

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