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.
Customers are using a variety of targeted gene editing methods such as ZFNs, TALENS and CRISPR/Cas9.
When choosing which cell line to edit, a key consideration is the need for a clonally pure population at the end of the process.
None of the currently available editing platforms have efficiencies that are high enough to guarantee a pool-derived, pure population of cells. Each platform has some potential for error, so it makes sense to compare more than one clonally derived cell line to ensure 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 current precision gene editing technique is the CRISPR/Cas9 system.
Following transfection and selection, the cells will need to undergo single cell cloning followed by expansion.
CRISPR/Cas9 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.
Single Cell Seeding
To obtain a stable cell line with the gene product eliminated, single clone cloning and isolation will rid the population of cells in which the gene is either incompletely knocked out or is untransfected, and which carry unwanted background mutations.
Due to low transfection efficiencies, for gene knock-outs single cell cloning will typically require a few hundred clones to be plated. For knock-ins, these occur at much lower frequencies, and may need over 1000 clones per edit.
Solentim has developed the VIPS as the preferred approach for single cell seeding. The VIPS will generate a resultant plate with high seeding efficiencies in around 10 minutes and due to the gentle nature of the dispensing method, most of these wells survive and grow up successfully into colonies.
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 essential for drawing any solid conclusions correlating a given mutation and cellular behaviour.
Expansion and Assurance of Clonality
After seeding single cells into 96 well plates, cells can be allowed to passively settle to the bottom of the well or can be centrifuged.
Imaging is continued for subsequent days of growth and ultimately colony formation. This can be 10-21 days depending on cell types.
A library of images is created for each well and it is possible to track back in time from the colony all the way back to Day 0 and confirm whether it started from a single cell or not.
Using Next Generation DNA sequencing (NGS) and bar-coded primers, it is possible to pool a sample of cells from the wells rather than process them independently. Once the correct mutation has been sequenced and identified, it can be easily deconvoluted and tracked back to which well/plate this clone came from.
A report can be rapidly generated for the well history of the top clones. This documentation package is called the Clonality Report and is importantly something which customers can use to support their clonality claims
Screening of Clones for Gene Edits
After using our systems to generate and document the single cell clones, customers will screen clones for the desired gene edit using a variety of techniques, which could include:
- PCR - amplifying the target site and looking for band shifts or loss/gain of a PCR product resulting from loss/gain of a primer site.
- SURVEYOR Assay - this is a gold-standard assay for screening for heterozygous clones (knock-outs or knock-ins)
- Restriction digest assay (RFLP) - if the targeting strategy is designed in such a way that a restriction site is lost or gained, this can be used to screen for targeted clones.
- Sequencing – simple Sanger sequencing or multiplexed NGS
- Immunoblots or Western blotting – absence of the protein can be confirmed for gene knock-outs