April 15, 2014 (Vol. 34, No. 8)

Enabling Gene Editing for Everyone with CRISPR and rAAV

With the emergence of new genome editing technologies such as CRISPR/Cas9, mammalian gene editing has come within the reach of the vast majority of skilled scientists. But undertaking a gene editing experiment is still fraught with significant challenges and even the most skilled scientists can benefit from proper planning and support to ensure that their gene editing experiments have the best chance of success.

The CRISPR system consists of two principal components: the Cas9 protein responsible for cleaving the genomic DNA and a guide RNA (gRNA) responsible for targeting Cas9 to a specific location within the genome (Figure 1). While the CRISPR system may seem simple on the surface, we caution researchers that there are multiple complexities beyond simply gRNA design involved in generating a suitable engineered stable cell line.

For demonstration purposes, let’s make the assumption that you know exactly what kind of modification you want to make to your cell line of choice. You’d like to introduce a point mutation into the coding region of a middle exon of your gene of interest. Here are a few of the complexities that need to be taken into account before heading directly into gRNA design:

How many copies of the target gene are there in your cell line of choice and do you need to edit all the alleles?
Targeting multiple alleles and multiple edits requires additional planning.

Do you know the exact sequence of the gene in your cells?
A single base mismatch can be the difference between success and failure when using CRISPR.

How do you plan to deliver the reagents into your cells? Do they transfect or electroporate well?
You can’t edit if the reagents aren’t efficiently delivered into your cells. Would a viral delivery system be better suited?

How critical is the risk of off-target modifications? Are you looking for a quick knock-out answer or do you need to be sure other parts of the genome aren’t affected?
There is a variant of Cas9 that only cuts a single strand of DNA and is less likely to introduce off-target modifications, but requires two appropriately spaced gRNAs to maintain a high level of targeting efficiency.

If you’re looking to introduce a SNP mutation, do you know the best way to design a donor that will improve your chances of generating the desired change without introducing other complicating modifications?
Donor strategy is a key part of any editing experiment designed to introduce specific genomic changes.

As a company focused on enabling translational genomics with many years’ experience in genome editing and custom cell line generation, Horizon has an extensive team of scientists dedicated to understanding the nuances of genome editing. This has allowed us to develop our new GENASSIST product offering so that it delivers the broadest possible range of software tools, services, and expert support.

Figure 1. Horizon Discovery’s GENASSIST™ range of gene-editing kits and reagents can help researchers generate their own CRISPR-ready cell lines that constitutively express Cas9-nickase. Use of wild-type Cas9 has been shown to lead to off-target cleavage, but a modified version introduces only single strand “nicks” that can be exploited to introduce subtle genetic changes while avoiding off-target effects common to double-strand breaks.

Case Study

A principal investigator approached Horizon seeking support with a knockout project in a human cell line. Upon discussing the intended use of the cell line in further research, we understood that he needed to obtain a complete functional knockout of two separate genes, complicated by the need to generate these modifications in a triploid line.

Horizon’s deep gene editing expertise allows us to shrewdly evaluate the needs of individual projects, and by being technology agnostic we are able to honestly discuss the benefits and limitations of each technical approach and recommend the optimal course of action.

While AAV-mediated genome editing, which Horizon is exclusively able to provide alongside CRISPR, is the most precise way to alter genomic DNA, it is not always the right choice. When multi-allelic knockouts are desired, nuclease-based approaches are often more suitable. In this case we proposed CRISPR technology, in particular Cas9 wild type because of the challenges of a triploid cell line and lack of concerns about off target effects from the scientist for this specific project.

In order to improve the odds of gene editing projects delivering the cell lines desired, Horizon has partnered with Desktop Genetics to develop a guide RNA design tool that combines the best of public models with Horizon’s internal expertise. We used this tool to identify five promising gRNAs for each of the two target genes.

It is now generally recognized that in order to have confidence that you are working with a suitably active gRNA, it’s best to design at least five gRNAs per target and test them in cells to make sure you obtain at least one with sufficiently high activity. We therefore validated each guide in K562 cells through use of a Surveyor® assay (Figure 2). The best performing guides were then provided to the scientist cloned into a backbone that expresses Cas9, together with a complete report on the guide design, performance, and position relative to the target sequence.

Although the scientist was not concerned about off-target effect for this particular project, we always recommend identifying likely off-target sites for unwanted gene editing events, and so we provide a list of possible off-target sites as part of the report.

In addition to guide RNA design and validation, Horizon offers donor design for knockins when an insertion or highly precise modification is required; cell lines that constitutively express Cas9 and are insertion-ready through a landing pad system, along with the means to generate further lines of this type;  our entire X-MAN™ library of over 550 isogenic cell lines that are available for further genome editing; access to our expert scientists for project planning and troubleshooting; and an extensive plasmid library.

Figure 2. In collaboration with Desktop Genetics, Horizon Discovery is developing a CRISPR design platform to quickly identify the best guide RNAs in the human genome for specific gene-editing tasks. Guide RNAs are validated by means of the Surveyor assay.

Eric Rhodes ([email protected]) is chief technology officer at Horizon Discovery.

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