Gene Editing With CRISPR/Cas: The CellCelector as a Perfect Tool for Targeted Cell Isolation and Single Cell Cloning
CRISPR-Cas9 genome editing is a method to genetically modify cells. For that purpose, cells are transfected with the Cas9 nuclease, an enzyme nonspecifically cutting DNA, and a synthetic guide RNA guiding the Cas9 nuclease to the DNA position, which shall be cut. The resulting double strand break activates cell intrinsic repair systems. By adding a repair template, homology directed repair (HDR) can insert sequences during the repair resulting in knock-in mutations. In absence of a repair template, repair is realized by non-homologous end joining (NHEJ). Though, NHEJ often causes small deletions or insertions interrupting the sequence of the repaired gene, resulting in knock-out mutations.
However, the outcome of CRISPR-Cas9 editing may differ among cells resulting in a heterogeneous cell population. In many experimental settings, it is necessary that cells have a homogenous genetic background. This can be achieved by single cell cloning of cells bearing the wished genotype.
The CellCelector represents the perfect tool for this application. The new single cell cloning method HT-NIC, developed in cooperation with the Probiogen AG, offers several advantages compared to commonly used cloning methods like limiting dilution cloning which include a single cell dilution step, so that cells are completely separated from each other to provide monoclonality. However, many cells might not tolerate being single-cell diluted leading to a high loss of cells because of the lack of cell-cell communication. With the new method this problem was solved. Significant cost and time savings, as well as an integrated monoclonality and viability proof, add to the attractiveness of the HT-NIC method.