“Time is of the essence” is a phrase that applies in many walks of life and certainly has applicability in the field of scientific research. Whether one is a graduate student preparing a critical thesis experiment or a pharma scientist developing the next blockbuster drug, getting to the key results quickly is critical.
Recently, a principal investigator approached Horizon Discovery looking to utilize genome editing technology to drive quick results with a knockout project in a human cell line. Upon discussing the intended use of the cell line in his research, it was understood that he needed to obtain a complete functional knockout of not just one, but two separate genes. The situation was further complicated by the need to generate these modifications in a triploid cell line.
The first step in any gene editing project should be to consider all of the main technologies available, and which would be best suited to that particular situation. Whilst homologous recombination with AAV-mediated genome editing is the most precise way to alter genomic DNA, there are also many benefits to nuclease-based approaches like ZFNs, TALENs, and CRISPR. Horizon Discovery has therefore adopted a “technology agnostic” approach to ensure that the technology best suited to the situation is applied, without undue allegiance to any one technique.
In this situation, where a triploid double gene knockout was required, a nuclease-based approach was deemed the most appropriate path forward, and CRISPR was recommended. CRISPR is a preferred method for multi-allelic modification due to the fact that fewer components are required to be transferred into the cells simultaneously than with a ZFN or TALEN approach. Because the investigator had limited concerns with potential off-target effects we recommended the use of the Cas9 wild-type protein in combination with appropriately designed guide RNAs targeting early conserved exons. An alternative approach involves the use of “Cas9-nickase”, a version of Cas9 that has a D10A mutation so that it only cuts a single strand. By using this form of Cas9 with two guide RNAs targeting opposite strands of the target DNA, a double-strand break is achieved with reduced off-target activity.
In order to improve the odds of success of gene editing projects using CRISPR, Horizon has partnered with Desktop Genetics to develop gUIDEbook™, a guide RNA design tool that combines the best public models with Horizon’s internal expertise. This tool was used to identify the five most promising gRNAs for each of the two target genes. Potential off-target sites for unwanted gene editing events are identified as part of the in silico design process, and efforts made to choose gRNA designs which limit potential off-target coding region disruptions.
It is generally recognized that in order to have confidence that you are working with a suitably active gRNA, it is best to design at least five gRNAs per target and test them empirically in cells to be sure of obtaining at least one with sufficiently high activity. This information about how each guide performed is fed into the gUIDEbook™ design algorithm, utilizing machine learning to create an ever-improving algorithm able to more accurately predict the highest activity guides.