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.