Magnetic Separation and Antibiotics Selection Enable Enrichment of Cells with ZFN/TALEN-Induced Mutations

Abstract

The ability to enrich cells with targeted mutations greatly facilitates the process of using engineered nucleases, including zinc-finger nucleases and transcription activator-like effector nucleases, to construct such cells. We previously used surrogate reporters to enrich cells containing nuclease-induced mutations via flow cytometry. This method is, however, limited by the availability of flow cytometers. Furthermore, sorted cells occasionally fail to form colonies after exposure to a strong laser and hydrostatic pressure. Here we describe two different types of novel reporters that enable mutant cell enrichment without the use of flow cytometers. We designed reporters that express H-2Kk, a surface antigen, and the hygromycin resistance protein (HygroR), respectively, when insertions or deletions are generated at the target sequences by the activity of engineered nucleases. After cotransfection of these reporters and the engineered nuclease-encoding plasmids, H-2Kk- and HygroR-expressing cells were isolated using magnetic separation and hygromycin treatment, respectively. We found that mutant cells were drastically enriched in the isolated cells, suggesting that these two reporters enable efficient enrichment of mutants. We propose that these two reporters will greatly facilitate the use of engineered nucleases in a wider range of biomedical research.

A stable but reversible integrated surrogate reporter for assaying CRISPR/Cas9-stimulated homology-directed repair

“…Consistent with this, we always detect mutations by T7E1 assays in mutated cells, whereas previous descriptions of episomal reporters often could not detect mutations in unselected cells (41,43,46). In such instances where the mutation load in unselected cells is undetectable by T7E1 assay (41,43,46), the -fold changes were calculated to be quite high, based on a denominator that is estimated (i.e. a mutation frequency of 0.5-1%).…”

Enhanced gene disruption by programmable nucleases delivered by a minicircle vector

“…To compare the activities of programmable nucleases expressed by minicircles versus conventional plasmids, we used pcDNA 3.0 ( Figure 1a) instead of the minicircle parental plasmid (Figure 1b) as the control vector because pcDNA 3.0 has been the primary vector used for programmable nuclease expression; [50][51][52] furthermore, the minicircle parental plasmid has not been used as an expression vector for programmable nucleases. Electrophoresis of the isolated minicircle vector showed that a major band of minicircle and a minor band of parental plasmid was present (Figure 1c).…”

“…[51][52][53][54][55][56] The reporter consists of the mRFP gene, followed by the nuclease's target sequence and the enhanced green fluorescent protein (eGFP) gene, which is out of frame with the mRFP gene ( Figure 2a). If ZFNs or TALENs make a double-strand break at the target sequence of the reporter, frame shifts occur because of the generation of indels in the target sequence through the error-prone non-homologous-end-joining repair process, resulting in functional eGFP expression.…”

“…The level of eGFP expression correlates with the activity of the programmable nucleases. [51][52][53][54] Flow cytometry was performed at 72 h after the cotransfection of the reporter plasmid and M-ZFN or P-ZFN into 293T cells. The frequency of eGFP + cells relative to transfected cells (eGFP + /total mRFP + cells) was significantly higher, by 1.71-fold, in the M-ZFN-transfected cell population than in the P-ZFN group (Figures 2b and c), suggesting that the ZFN activity is higher when the enzyme is delivered by a minicircle vector versus a conventional plasmid.…”

“…51 For the reporter assay, untransfected cells and cells transfected with reporters alone were used as analysis controls. [51][52][53] For the viability assay, unstained cells and singlecolor stained cells were used as analysis controls. In each sample, 20 000 cells were analyzed.…”