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Genome engineering using Cas9/gRNA Ribonucleoproteins (RNPs)

Posted by Joel McDade on Apr 21, 2016 10:30:00 AM

CRISPR has quickly become the preferred system for genome engineering due to its simplicity, as it requires only Cas9 and a guide RNA (gRNA).  Choosing the correct method to deliver both Cas9 and gRNAs to your target cells is absolutely critical as failure to adequately express either component will result in a failed experiment.  In our previous blog post entitled “CRISPR 101 - Mammalian Expression Systems and Delivery Methods” we provided a general overview of the most common ways in which you can deliver Cas9 and gRNAs to your target cells and discussed a few key advantages and disadvantages of each method. In this blog post, we will go into greater detail about why and how Cas9/gRNA Ribonucleoprotein complexes (Cas9 RNPs) are being used for genome engineering experiments and provide a general framework for getting started with Cas9 RNPs in your research.

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Topics: CRISPR, CRISPR Expression Systems and Delivery Methods

Optimizing Donor DNA for Enhanced CRISPR Genome Editing

Posted by Guest Blogger on Mar 24, 2016 10:30:00 AM

This post was contributed by guest blogger Chris Richardson, a Postdoctoral Researcher in Jacob Corn’s lab.

CRISPR-Cas9 (Cas9) is an RNA-guided nuclease that targets and cuts genomic DNA. The interplay between Cas9 (which causes the breaks) and host cell DNA repair factors (which repair those breaks) makes Cas9 extremely effective as a genome editing reagent. This interplay falls into two broad categories and thus, causes two types of editing outcomes: Cas9 breaks repaired by the non-homologous end-joining (NHEJ) pathway disrupt target gene sequences (thus inactivating genes), while breaks repaired by homology directed repair (HDR) pathways can modify the sequence of a gene (thus altering its function). HDR is crucial for certain applications, for example, correcting the allele that causes sickle cell anemia. However, HDR occurs much less frequently than NHEJ and the efficiency of these editing reactions is low. Understanding the biological cause of this repair bias is a fascinating (and yet unanswered) question. Our recent paper (Richardson et al 2016) revealed some of the biophysical parameters that can influence the HDR/NHEJ decision.

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Topics: CRISPR, CRISPR Expression Systems and Delivery Methods

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: CRISPR, CRISPR Expression Systems and Delivery Methods

CRISPR 101: Mammalian Expression Systems and Delivery Methods

Posted by Nicole Waxmonsky on Sep 24, 2015 10:30:00 AM

This post was updated on Dec 4, 2017.

CRISPR technology has been widely adopted for genome editing purposes because it's cheaper, faster, and easier than prior editing techniques. More and more CRISPR tools are being published each month, making CRISPR a great choice for your next experiment!

In this blog post we’ll provide an overview of some CRISPR mammalian expression systems, the typical applications for each, and potential delivery methods.

 

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Topics: CRISPR, CRISPR 101, CRISPR Expression Systems and Delivery Methods

A Match Made in Heaven: CRISPR and AAV

Posted by Mary Gearing on Jul 14, 2015 10:30:00 AM

This post was updated on Dec 4, 2017.

CRISPR genome editing has quickly become the most 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 chimeric sgRNA together (~4.2 kb) into an AAV vector is challenging due to the low packaging capacity of AAV (~4.5 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

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