Dual-reporter surrogate systems for efficient enrichment of genetically modified cells

Abstract

Isolation of genetically modified cells generated by designed nucleases are challenging, since they are often phenotypically indistinguishable from their parental cells. To efficiently enrich genetically modified cells, we developed two dual-reporter surrogate systems, namely NHEJ-RPG and SSA-RPG based on NHEJ and SSA repair mechanisms, respectively. Repair and enrichment efficiencies of these two systems were compared using different nucleases. In both CRISPR-Cas9- and ZFNs-induced DSB repair studies, we found that the efficiency and sensitivity of the SSA-RPG reporter with direct repeat length more than 200 bp were much higher than the NHEJ-RPG reporter. By utilizing the SSA-RPG reporter, we achieved the enrichment for indels in several endogenous loci with 6.3- to 34.8-fold of non-selected cells. Thus, the highly sensitive SSA-RPG reporter can be used for activity validation of designed nucleases and efficient enrichment of genetically modified cells. Besides, our systems offer alternative enrichment choices either by puromycin selection or FACS.

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%).…”

“…Of note, all of the episomal reporters share one limitation: they rely on plasmid DNA transfected into cells where it exists transiently as an extra-chromosomal fragment. The use of transient episomal DNA likely limits the utility of such surrogate reporter assays to cell lines that can be transfected with high efficiency, such as HEK293 cells, a cell type frequently used for demonstrating the efficacy of episomal reporters (41)(42)(43)(44). Furthermore, transient episomal reporters that confer resistance to antibiotics such as hygromycin or puromycin are cumbersome for achieving optimal enrichment of modified cells before loss of the episomal vector; the timing and efficacy of such selection is dubious because the precise moment of episomal loss is unpredictable, which complicates antibiotic treatments that require Ͼ3-4 days of selection.…”

Multiplex CRISPR/Cas9-based genome engineering enhanced by Drosha-mediated sgRNA-shRNA structure

“…The corresponding RPG surrogate reporter vectors (pRPG, Fig. 3b) were constructed as we previously reported1723 and used to facilitate the genome targeting assays. The HEK293T cells were preliminarily co-transfected with the msgRNA-3 construct, hStCas9 expression vectors together with the pRPG.AAVS1 vector.…”

“…Although it has been well accepted that in mammalian cells DSBs are primarily repaired by the non-homologous end joining (NHEJ) repair mechanism2021, SSA may be more frequent if substantial repetitive elements are proximal to the break ends22. As previously reported, we have developed a series of surrogate reporters based on the SSA-mediated precise repair of reporter genes for evaluating the activity of artificial site-specific nucleases or enriching genetically modified cells172324.…”

“…To improve the targeting efficiency, we used the same RPG (DsRed-Puro r -eGFP) surrogate reporter system as previously reported1723 to aid the enrichment of positive cells with genomic modification as well as the selection of positive cell clones. When the nuclease functions on the RPG reporter, the interrupted puromycin resistant gene ( Puro r ) can be repaired through the SSA repair pathway precisely.…”