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Mary Gearing

Mary Gearing is a Scientist at Addgene. She got her start as a Science Communications Intern writing for the Addgene blog and website. As a full-time Addgenie, she still enjoys blogging about CRISPR and other cool plasmids!

Recent Posts

CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

Posted by Mary Gearing on Sep 28, 2020 8:00:00 AM

Originally published Mar 3, 2016 and last updated Sep 28, 2020 by Will Arnold.

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 use CRISPR  in E. coli and other 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: CRISPR, CRISPR Expression Systems and Delivery Methods

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

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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CRISPR 101: Cytosine and Adenine Base Editors

Posted by Mary Gearing on Aug 6, 2020 10:30:00 AM

Originally published Aug 16, 2016 and last updated Aug 6, 2020 by Jennifer Tsang.

When we talk about CRISPR applications, one negative often comes up: the low editing efficiency of homology-directed repair (HDR). Compared to non-homologous end joining, HDR occurs at a relatively low frequency, and in nondividing cells, this pathway is further downregulated. Rather than try to improve HDR, scientists have developed two classes of base editors: cytosine base editors (CBEs) and adenine base editors (ABEs). 

(There are also RNA base editors, but we’ll just be covering DNA base editors here. To learn more about RNA base editors head over to this blog post: CRISPR 101: RNA Editing with Cas13)

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Topics: CRISPR, Base Editing

CRISPR 101: RNA Editing with Cas13

Posted by Mary Gearing on Jul 31, 2020 8:30:00 AM

Originally published Nov 30, 2017 and updated Jul 31, 2020.

Cas13 enzymes are quickly becoming major players in the CRISPR field. Just a year after Feng Zhang’s lab identified Cas13a (C2c2) (Abudayyeh et al., 2016) as a RNA-targeting CRISPR enzyme, they adapted Cas13b for precise RNA editing (Cox et al., 2017). This new system, termed REPAIR (RNA editing for programmable A to I (G) replacement) is the first CRISPR tool for RNA editing. Two years after that, the lab published a paper on an RNA editor that allows C to U edits (RESCUE). We’ll walk through how these tools were developed and potential ways you can use it in your research.

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Topics: CRISPR, CRISPR 101, Cas Proteins

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