During the past five years, the use of genome modification tools has seen an extraordinary gain in momentum after the discovery of the ground-breaking CRISPR/CAS9 technology.
There are also technologies alternative to CRISPR, such as TALEN and zinc-finger nucleases (ZFN) that similarly facilitate the manipulation of the cellular DNA. Taken together they all enable the possibility to generate genome modifications in vitro relatively easily. Therefore, the field of discovery biology has entered a new era of more relevant molecular and phenotypic observations. Indeed, prior to the advent of genome editing, the modulation of cellular pathways was quite exclusively limited to mRNA-targeted agents and/or chemical compounds.
Genome editing has proved useful to test molecular and cellular biology hypotheses, especially when the complete elimination/modification of the endogenous molecule is required to obtain the desired phenotype. In addition, this approach is scalable, and has been coupled with recent improvements in single-cell sequencing technologies. Therefore, genome editing techniques are excellent tools for genetic screens aimed at identifying key elements in disease phenotypes and/or synthetic lethality.
The ability to knock-out, modify, tag and alter endogenous proteins has generated a number of different efforts in the field that are not always backed by a strong genetics expertise. They might therefore lead to inaccurate conclusions.
Our experience in genetics and genome modification has allowed us to generate a path for genetic modifications in discovery biology studies that provides robust results:
- Profiling of the genetic background of the receiving cell type (including karyotype and array-CGH).
- Sequencing of target region(s)
- Genotyping strategy
- Use of recombinase cassette exchange when multiple variants are desired
- Use of safe harbors for exogenous sequence integration
- Generation of isogenic cell lines
- Sequencing of the final clones
Through genome editing, we were able to obtain several type of modifications: from simple targeted mutations to tagging with large reported proteins, and even complete deletion of big genomic regions of about 200 kbp.