Articles, announcements and insights from Horizon Discovery

Optimization of CRISPR Delivery in Cultured Cells & Single Cell Embryos

Jun 13, 2016 10:22:03 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

Frequently Asked Questions For HAP1 Knockout Cell Lines

Jun 10, 2016 1:29:00 PM No Comments
Here you'll find a complete list of all our most frequently asked questions relating to HAP1 knockout cell lines. If you want to know how they're generated, how they're validated or how to find out if they're right for you - this is a great place to start.
Read More Cell lines

Frequently asked questions for X-MAN Cell Lines

Jun 10, 2016 1:27:59 PM No Comments

Here you'll find a complete list of our most frequently asked questions relating to X-MAN cell lines. How are they made, how are they validated, and how can you use them? Read on to find out.

Read More Cell lines

[Protocol] How to bank cell lines

Jun 10, 2016 1:26:45 PM No Comments

Whether it's a stock you've expanded following it's arrival from the cell bank, or a clone you've carefully nurtured from the single cell, banking down your cells in the right way is crucial if you're going to be able to return to them time and again, and revive them quickly so that you can get on with your experiments.

Here's our protocol for banking human cell lines:

Read More Cell lines, Protocols

[Protocol] Optimizing transfection for genome editing

Jun 10, 2016 1:25:54 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 1:25:18 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 1:23:34 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

Rapid identification of potential drug targets using endogenous reporter cell lines and siRNA screening

Jun 10, 2016 1:22:13 PM No Comments

We have combined our proprietary genome editing technology with large scale RNAi screening capabilities to allow identification of novel drug targets.

Read More Cell lines, siRNA Screening, Endogenous reporters

Developing Cellular Reporter Technologies Using Horizon's Cell Models

Jun 10, 2016 1:20:39 PM No Comments

Isogenic cell lines provide genetically defined, patient-relevant, predictive in vitro models of genetic disease. We are further extending their application within targeted drug discovery with the development of new reporter disease models using our gene engineering technology. These models combine Horizon's cell lines with the endogenous gene reporting capabilities in the form of NanoLuc® luciferase and HaloTag® reporter technologies.

Read More Cell lines

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

Jun 10, 2016 1:10:39 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

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