Cell Engineering and Precision Gene Editing

CRISPR

This technology is now commonly employed by drug discovery groups making cell lines for in vitro disease models. These can then be used for target discovery and validation. Customers are using a variety of targeted gene editing methods such as ZFNs, TALENS and CRISPR/Cas9.

Cell Engineering

One very important consideration for choosing a particular cell line to edit has to do with the need to obtain a clonally pure population at the end. None of the editing platforms has efficiencies high enough to guarantee a pure population of cells derived from a pool. In addition, each platform has some degree of off-target potential and it is generally a good policy to obtain more than one clonally derived cell line to compare to make sure any phenotypes observed are due to the targeted change and not some opportunistic off-target effect or other component of genetic drift.

The most popular precision gene editing technique is the CRISPR/Cas9 system (see workflow diagram above) which consists of two components: a Cas9 protein with endonuclease activity, and a guide RNA (gRNA) that confers specificity to the system by sequence-dependent recognition. After binding of the gRNA to the targeted DNA, the endonuclease activity of Cas9 generates a double-stranded break at the targeted genomic DNA site.  At this point, researchers can take advantage of the cell’s DNA repair mechanisms to fix the double-stranded break. If the objective is to simply knock-out expression of the target gene, the cell’s non-homologous end joining (NHEJ) pathway can be exploited.

This DNA repair pathway is error-prone by nature, as it often includes or deletes nucleotides at the site of the break during the end-joining process. The loss of nucleotides invariably interrupts the open reading frame – often resulting in the generation of a premature stop codon – which prevents the protein from being produced.

If a more precise edit of the gene is necessary, the homology-driven repair pathway (HDR) can be employed. By designing a repair template containing the desired sequence change, researchers can use homologous recombination to introduce (or knock-in) any alteration into the genomic DNA.

In the workflow, following transfection and selection, the cells will need to undergo single cell cloning followed by expansion. To obtain a stable cell line with the gene product completely eliminated, single clone cloning and isolation will rid the population of cells in which the gene is either incompletely knocked out or that went untransfected, and that carry unwanted background mutations. Clonal isolation is followed by an expansion period to establish a new clonal cell line.

Pure clonal isolation from a single progenitor cell is a critical step in the genetic and functional characterization of mutations achieved by the CRISPR/Cas9 system. While traditionally it can be the most laborious and time-consuming step in CRISPR-based genome engineering using cell models, generating clonal mutant cell lines is absolutely required to draw any solid conclusions correlating a given mutation and cellular behaviour.

CRISPR/Cas9 application in Drug Discovery

These groups will typically be making cell lines as reagents containing knock-outs (deletions) or knock-ins (insertions) for a gene of interest to generate disease phenotypes in vitro and to use these for validating drug targets.

Cell types can vary from a range of immortalised (cancer) cells from different tissues, to CHO, HEK and iPSCs.

The technique is particularly helpful in generating isogenic cell lines (matched pair of cells which differ only in the mutation) for in vitro target discovery and validation.

It should be noted that CRISPR/Cas9 may have future therapeutic applications, but currently is largely limited to research and discovery due to a complex intellectual property landscape which surrounds the area.

Next Steps