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CRISPR Activators: A Comparison Between dCas9-VP64, SAM, SunTag, VPR, and More!

Posted by Gabrielle Clouse on Oct 6, 2020 9:15:00 AM

Prior to the discovery of CRISPR/Cas systems, gene activation across multiple loci was an arduous process. When using zinc finger proteins or TALE proteins, proteins had to be re-engineered for each gene, making wide-scale gene activation seem next to impossible. The development of CRISPR/Cas systems, however, greatly improved the simplicity of gene activation: rather than requiring protein engineering for each loci, CRISPR/Cas systems only require changing the programmable guide RNA.

Gene activation by dCas9, also referred to as CRISPRa, was initially published in 2013 (Bikard et al., 2013, Perez-Pinera et al., 2013). In the years that followed, innovative methods greatly improved CRISPRa, expanding its practicality and popularity in research (Tanenbaum et al., 2014, Konermann et al., 2015, Chavez et al., 2015).

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Topics: CRISPR

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

How to Design Your gRNA for CRISPR Genome Editing

Posted by Guest Blogger on Sep 24, 2020 9:15:00 AM

Originally published May 3, 2017 and last updated Sep 24, 2020

This post was contributed by guest blogger, Addgene Advisory Board member, and Institute Scientist at the Broad Institute, John Doench.

CRISPR technology has made it easier than ever both to engineer specific DNA edits and to perform functional screens to identify genes involved in a phenotype of interest. This blog post will discuss differences between these approaches, and provide updates on how best to design gRNAs. You can also find validated gRNAs for your next experiment in Addgene's Validated gRNA Sequence Datatable. A more extended discussion of these subjects can be found in two recent review articles (Doench et al., 2017, and Hanna et al., 2020) and references therein.

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Topics: CRISPR, CRISPR gRNAs

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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Topics: CRISPR, CRISPR 101, CRISPR gRNAs

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