Articles, announcements and insights from Horizon Discovery

Off-target editing with CRISPR/Cas9 : The evidence

Jun 23, 2017 10:16:36 AM No Comments

What more do we know since last year?

As more and more papers are published using models generated by CRISPR/Cas9 editing, and new and exciting applications for the CRISPR/Cas system continue to be invented, the potential for off-target editing continues to be discussed. We published an article on our blog a year ago which explains the potential for off-target editing with CRISPR/Cas9 and summarised some of the literature on this topic, and thought this was a good time for an update.

 
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How to Cure Cancer in 3 Easy Steps

Mar 6, 2017 6:10:15 PM No Comments

When I have read articles just like this one early on in my career, I would laugh and categorize it with blogs regarding Bigfoot and the Loch Ness Monster. However, much has changed in the past 10 years. New technologies have been developed and milestones have been reached that should have Cancer a little worried. These 3 steps might be viewed to some as obvious, but I argue that it’s how the researchers have utilized the technology wisely that has made the difference. I have identified some papers that have carved a successful path to Cancer's possible demise.

Read More Cell lines, Gene editing, CRISPR, Oncology Panels, Target identification, Target validation, PDX, Cancer models, Preclinical efficay testing, High Fidelity Models

Should I be worried about CRISPR off-target effects?

Jun 17, 2016 11:06:42 AM No Comments

Increasingly the literature suggests that CRISPR's potential for off-target effects is not as “bad” as originally thought. Here are a few things to set your mind at ease if you’re working with the CRISPR-Cas9 system:

Read More Gene editing, CRISPR

5 tips for guide RNA design

Jun 15, 2016 4:24:37 PM No Comments

Early on in our CRISPR use at Horizon, we learned that that when it comes to guide RNA design not all sgRNAs are created equal. In fact what makes a good guide is still the topic of a great deal of research in the field – and aside of the consensus that a guide with a guanine at position -1 is more likely to have a higher activity, we are still a way from being 100% confident in predicting in silico guide activity in vitro/vivo.

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Optimization of CRISPR Delivery in Cultured Cells & Single Cell Embryos

Jun 13, 2016 11:22:03 AM No Comments

The CRISPR/Cas9 system has been rapidly adapted to practically every model system for its ease to generate and high efficiencies to cleave target DNA. But unlike our experience with Zinc Finger Nucleases, in the human, rat and mouse cell lines we tried successful co-transfection of Cas9 mRNA and sgRNA was cell-line dependent, and often resulted in either very low or no cleavage activities.

However, sequential transfection of cells with Cas9 DNA first, and sgRNA followed 24 hrs later, reliably produced good level of activity, indicating the requirement of Cas9 presence at the time of introduction of sgRNA. Not surprisingly, creation of a cell line stably expressing Cas9 led to consistently high cleavage activities upon transfection of sgRNAs. Transfection of recombinant Cas9 protein pre-complexed with sgRNA (ribonucleoprotein particles, or RNPs) led to efficient cleavage as well.

On the other hand, when Cas9 mRNA and sgRNAs are co-microinjected into single cell embryos, it produces target cleavage as efficiently as RNPs to produce straight KOs and large deletions between two target sites, again raising a question of local concentrations of Cas9 protein and sgRNA.

Below we summarize some of the work we've done optimizing delivery of CRISPR-Cas9, which which can be read in full publication form in Human Gene Therapy here.

Read More Cell lines, Gene editing, Microinjection

[Protocol] Optimizing transfection for genome editing

Jun 10, 2016 2:25:54 PM No Comments

Transfection or electroporation is used to efficiently introduce the nucleic acids required for CRISPR cell line engineering into a cell line. The type and number of nucleic acids (usually plasmids) being introduced into a cell line will depend on the engineering event being undertaken. At its most simple, gene knockouts can be achieved by transfection of a plasmid expressing wild-type Cas9 along with a guide RNA (gRNA) to the gene that is being knocked out.

Read More Cell lines, Gene editing, Protocols

[Protocol] Harvesting and analyzing clones following CRISPR gene editing

Jun 10, 2016 2:25:18 PM No Comments

If you've followed our guide to planning a successful genome editing experiment, then you'll hopefully be working in optimal conditions, and have a good idea of your guide's editing efficiency. This number should give you a rough idea of how many clones you're going to have to screen to find a targeted clones. Keep in mind the following:

Read More Cell lines, Gene editing, Protocols

[Protocol] Expansion of cell line clones from single cells

Jun 10, 2016 2:23:34 PM No Comments

Once a clone has been identified positive for targeting in the initial screen, the cells should be expanded to a 48-well plate. Expansion of the cells can usually occur at 1-4 days post screen.

Read More Cell lines, Gene editing, Protocols

Planning a CRISPR gene editing experiment to maximize your chances of success

Jun 10, 2016 2:10:39 PM No Comments

While CRISPR-Cas9 has made gene editing cheaper, easier and more accessible than ever before, using the system can in some cases still be challenging, and no scientist can yet be 100% certain of success. With careful preparation and planning however, chances of success can be significantly boosted.

Read More Gene editing

Improving Gene Editing Success with An Optimised Cell Line Screening Procedure

Jun 10, 2016 2:06:25 PM No Comments

There exist now a range of techniques to perform genome editing, such as ZFN, CRISPR, TALENS and AAV, each with their own strengths and weaknesses. However, one consistent element that has a significant impact on the success of that editing event when generating an isogenic cell line is the choice of parental cell line to be engineered.

Read More Cell lines, Gene editing

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