Strategies for the Enrichment and Selection of Genetically Modified Cells

Plasmid encoded marker genes can be used for the enrichment of genetically modified cells by selecting transfection-positive cells, and surrogate reporters can be used for the enrichment of genetically modified cells by selecting nuclease-active cells. Endogenous marker genes not only indicate genetically modified cells directly but also can be used as a cotargeting strategy to enrich cells with targeted modifications at other unlinked loci of interest. Resection and annealing methods can enable efficient error-free targeted insertions, even in nondividing cells such as neurons. Although knockin selection markers are ideal for selecting precise integration-positive cells, further excision of the selection marker cassette is required. Alternative and more efficient strategies for enrichment and selection of genetically modified cells should be explored. Programmable artificial nucleases have transitioned over the past decade from ZFNs and TALENs to CRISPR/Cas systems, which have been ubiquitously used with great success to modify genomes. The efficiencies of knockout and knockin vary widely among distinct cell types and genomic loci and depend on the nuclease delivery and cleavage efficiencies. Moreover, genetically modified cells are almost phenotypically indistinguishable from normal counterparts, making screening and isolating positive cells rather challenging and time-consuming. To address this issue, we review several strategies for the enrichment and selection of genetically modified cells, including transfection-positive selection, nuclease-positive selection, genome-targeted positive selection, and knockin-positive selection, to provide a reference for future genome research and gene therapy studies. Programmable artificial nucleases have transitioned over the past decade from ZFNs and TALENs to CRISPR/Cas systems, which have been ubiquitously used with great success to modify genomes. The efficiencies of knockout and knockin vary widely among distinct cell types and genomic loci and depend on the nuclease delivery and cleavage efficiencies. Moreover, genetically modified cells are almost phenotypically indistinguishable from normal counterparts, making screening and isolating positive cells rather challenging and time-consuming. To address this issue, we review several strategies for the enrichment and selection of genetically modified cells, including transfection-positive selection, nuclease-positive selection, genome-targeted positive selection, and knockin-positive selection, to provide a reference for future genome research and gene therapy studies. a ‘self-cleaving’ small peptide that allows for coexpression of multiple genes from a single transcript, separated by peptide cleavage. a genome editing technology that can convert a specific DNA base into another at a targeted locus. a site-specific recombinase technology used to carry out deletions, insertions, translocations, and inversions at specific sites in the genome. a DNA-editing technology adapted from a naturally occurring genome editing system in bacteria. a DNA-editing technology analogous to the CRISPR/Cas9 system but originating from Prevotella and Francisella. marker genes supplied as donors and integrated into the genome for selection. one type of DNA damage that causes a break in both strands of the DNA helix. genes with specific characteristics in cells that are sensitive to corresponding treatments. a specialized type of flow cytometry used for cell counting, sorting, and biomarker detection. a naturally occurring nucleic acid repair system where a homologous template is supplied. a homology-independent targeted integration repair pathway that allows for robust knockin in both dividing and nondividing cells. a DNA repair pathway that uses long homologous sequences to align the broken strands before joining. a molecular biology term for an insertion or deletion of a small number of bases in a DNA sequence. a cell separation method based on magnetic isolation technology. a DNA repair pathway that uses 5–25 base pair microhomologous sequences to align the broken strands before joining. a modified version of Cas9 protein that cleaves DNA and results in single-strand breaks. a pathway that repairs double-strand breaks without the need for a homologous template. a mobile genetic element that efficiently transposes between vectors and chromosomes via a ‘cut and paste’ mechanism. selectable marker genes in a plasmid. a DNA repair pathway harnessed from MMEJ for gene knockin. single strand annealing of homologous repeat sequences that flank a DSB, which causes a deletion rearrangement between the repeats. contain the target sequence and faithfully reflect a nuclease’s activity on the chromosome in the same cell. a DNA repair pathway that uses a linearized dsDNA donor with long homologous sequences to integrate. artificial restriction enzymes generated by fusing a transcription activator-like effector DNA-binding domain to a DNA-cleavage domain, which are functional as dimers. artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain, which are functional as dimers.